Modified lantibiotics and methods of making and using the modified lantibiotics

ABSTRACT

The subject invention pertains to a modified lantibiotic containing an intact cysteine at the C-terminus, particularly, a cysteine that is not decarboxylated and that contains a free carboxyl group. Derivatives of the modified lantibiotic comprising a moiety conjugated to the carboxyl group of the terminal cysteine are also provided. A bacterium that produces a modified lantibiotic having an intact cysteine at the C-terminus are also provided, wherein the bacterium is genetically modified to inactivate a gene that encodes a decarboxylase enzyme that decarboxylates the cysteine at the C-terminus of a precursor lantibiotic. Methods of producing a modified lantibiotic having an intact cysteine at the C-terminus by culturing a bacterium that synthesizes the modified lantibiotic and purifying the lantibiotic are also provided. Mutants of lantibiotics, particularly, mutacin 1140 having higher anti-bacterial activity or higher bacterial expression compared to mutacin 1140 are also provided.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application Ser.No. 62/414,334, filed Oct. 28, 2016, the disclosure of which is herebyincorporated by reference in its entirety, including all figures, tablesand amino acid or nucleic acid sequences.

The Sequence Listing for this application is labeled“Seq-List-replace.txt” which was created on Oct. 2, 2019 and is 25 KB.The entire content of the sequence listing is incorporated herein byreference in its entirety.

BACKGROUND OF THE INVENTION

Lantibiotics are characterized by their post-translational modifications(PTMs). Dehydrations of serine and threonine residues intodehydroalanine and dehydrobutyrine residues, respectively, are commonmodifications found in lantibiotics. These dehydrated residues arecyclized with cysteines to form thioether bridges, which are calledlanthionines¹⁻². Lantibiotics can contain other post-translationalmodifications, such as D-alanines in lacticin 3147, β-hydroxy aspartatein cinnamycin, 2-oxopropionyl in lactocin S, and an oxidized lanthioninein actagardine³⁻⁶.

The epidermin group of lantibiotics and other lantibiotic peptides havea S—[(Z)-2-aminovinyl]-D-cysteine (AviCys) residue at the C-terminal endof the peptide (FIG. 1a ). This residue consists of a decarboxylatedcysteine which forms a lanthionine ring. Several of these modificationsare important for lantibiotic activity; however, the importance of theAviCys for the lantibiotic activity in the epidermin group oflantibiotics is not known.

Mutacin 1140, produced by Streptococcus mutans JH1140, is a lantibioticthat has shown promise as a potential therapeutic (FIG. 1b )⁷⁻⁹. It hasa broad spectrum of activity against Gram-positive bacteria¹⁰. Further,mutacin 1140 has been shown to clear Staphylococcus aureus infections inrodent models with little toxicity¹¹. The bacterium producing mutacin1140 has been engineered into a therapy for preventing dental caries¹².

Mutacin 1140 belongs to the class I epidermin group of lantibiotics andis structurally related to epidermin and gallidermin¹³⁻¹⁴. The first twolanthionine rings, rings A and B, of the epidermin group are referred toas the lipid II binding domain. The lantibiotic nisin shares structuralhomology to the lanthionine rings A&B. The latter half of the epiderminand nisin peptide is referred to as the lateral assembly domain, whichpresumably abducts lipid II into large lipid II/lantibioticcomplexes¹⁵⁻¹⁶.

Decarboxylation of a C-terminal cysteine to form an AviCys residueoccurs in several metabolites¹⁷. AviCys is present in the class IIlantibiotics mersacidin and microbisporicin¹⁷⁻¹⁸. It is also found innon-lantibiotics, such as cypemycin. Cypemycin contains many of thelantibiotic PTM modifications; however, it does not form lanthioninerings¹⁹. The AviCys residue has also been found in the nonribosomalpeptide synthetases (NRPS) produced metabolite thiovideramide. Themechanism of AviCys formation for the NRPS peptide maybe different duethe nature of its biosynthesis²⁰.

In certain lantibiotics, decarboxylation of cysteine at the C-terminusis performed by the flavoprotein LanD. This decarboxylase has been shownto be specific for C-terminal cysteines. Furthermore, LanD could notdecarboxylate an ethyl-thioether mimic, suggesting decarboxylationoccurs before ring D formation²¹. Crystal structures for both EpiD, thedecarboxylase for epidermin, and MrsD, the decarboxylase for mersacidin,indicate that these enzymes form a homo-dodecamer²²⁻²³. Studies on themechanism of activity suggest that decarboxylation produces an ene-thiolintermediate that promotes terminal ring formation²⁴. There have been noreports of an isolated carboxylated analog of an AviCys containinglantibiotic, even in an EpiD deletion mutant of epiderminbiosynthesis²⁵.

HOAt/EDC coupling has been achieved for lantibiotics that contain aC-terminal carboxyl group. NVB302, an analog of actagardine which hasundergone phase 1 clinical trials, has a diaminoheptane tail attached tothe C-terminus of the lantibiotic²⁷. Additionally, lantibiotics can beproduced through solid-phase peptide synthesis using orthogonallyprotected lanthionine rings^(26, 28).

BRIEF SUMMARY OF THE INVENTION

Chemical modification of lantibiotics offers a novel avenue for thedevelopment of new therapeutics²⁶. The lack of a C-terminal carboxylgroup complicates the further development of the lantibiotics containinga C-terminal cysteine, for example, epidermin group of lantibiotics,using C-terminal modifications. A free carboxyl group analog of thelantibiotics containing a C-terminal cysteine would promote studiesaimed at understanding the functional basis for AviCys residues withinthe lantibiotic and promote the synthesis of lantibiotics havingimproved therapeutic efficacy.

Accordingly, an embodiment of the invention provides a modifiedlantibiotic containing an intact cysteine at the C-terminus,particularly, a cysteine that is not decarboxylated and that contains afree carboxyl group. Derivatives of the modified lantibiotic comprisinga moiety conjugated to the carboxyl group of the terminal cysteine arealso provided. In certain embodiments, the moiety conjugated to thecarboxyl group of the modified lantibiotic having an intact C-terminalcysteine is a functional group or a detectable label.

A bacterium that produces a modified lantibiotic having an intactcysteine at the C-terminus is also provided, wherein the bacterium isgenetically modified to inactivate a gene that encodes a decarboxylaseenzyme that decarboxylates the cysteine at the C-terminus of a precursorlantibiotic.

Methods of producing a modified lantibiotic having an intact cysteine atthe C-terminus by culturing a bacterium that synthesizes the modifiedlantibiotic and purifying the lantibiotic are also provided.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication, withcolor drawing(s), will be provided by the Office upon request andpayment of the necessary fee.

FIGS. 1A-1B. Structures of class I and class II lantibiotics. (A)Structures of class I lantibiotics: mutacin 1140 (SEQ ID NO: 33),epidermin (SEQ ID NO: 34), and nisin (SEQ ID NO: 35). Dehydratedresidues are either Dha or Dhb. The lipid II binding domain of class Ilantibiotics consist of the first two lanthionine rings A and B, whilethe lateral assembly domain consist of the terminal rings. (B) Structureof the class II lantibiotic, mersacidin (SEQ ID NO: 36), which containsan AviCys. Residues involved in AviCys formation are labeled in red forboth classes.

FIG. 2. Scheme for chemical modification of mutacin 1140-COOH. Mu1140COOH analog was coupled to various primary amines using HOAt/EDCcoupling. Primary amines were chosen based on size and differences inphysiochemical properties. Reaction conditions were constant for eachsubstrate, and yields were greater than 80% for each substrate.

FIGS. 3A-3C. In vivo localization of mutacin 1140. B. Subtilis PY79cells were incubated with various fluorescein conjugated lantibiotics.(A) A no antibiotic sample was used as negative control. (B) Fluoresceinconjugated nisin binds and abducts lipid II to form patches as expected.(C) Fluorescein conjugated mutacin 1140 has a similar localizationpattern as nisin. Images were taken using a confocal Olympus microscopeusing a 100× objective, or a 40× objective for the control.

FIGS. 4A-4D. Lipid II competition assay of nisin and mu1140-COOH analog.B. subtilis cells were treated with mu1140-COOH to compete with nisin inbinding to lipid II. (A) Solvent blank (no antibiotic) control. (B)Fluorescein labeled nisin binds tightly to lipid II to form patches. (C)Prior treatment of cells with mu1140 prevents binding of fluoresceinlabeled nisin. (D) Prior treatment of cells with mu1140-COOH drasticallyreduces binding of fluorescein labeled nisin. Images were taken using aconfocal Olympus microscope using a 100× objective, or a 40× objectivefor the control.

FIGS. 5A-5C. Deletion of mutD in S. mutans JH1140. (A) Scheme for thedeletion of mutD by IFDC2 gene replacement. (B) Deferred antagonismassay against M. luteus ATCC 10240 shows no zone of inhibition foreither S mutans IFDC2:mutD or S. mutans ΔmutD. S. mutans JH1140 and S.mutans ΔmutA were used as a positive and negative controls,respectively. (C) Purification of S mutans IFDC2:mutD extracts (green)show a single peak, while there was no observable peak for S. mutansΔmutA and ΔmutD.

FIGS. 6A-6B. Edman sequencing of mutacin 1140-COOH. (A) After doublelabeling with sodium borohydride and ethanethiol, a thio-ethyl cysteine(S-EC) or beta-methyl thio-ethyl cysteine (BM-S-EC) is expected at sitesof lanthionine ring formation in the Edman sequence compared to mutacin1140 (SEQ ID NO: 37). Blue circles indicate residues expected to formlanthionine rings, and green circles indicate sites of dehydration. (B)Select Edman sequence (SEQ ID NO: 38) spectras for the modified residuesindicate full modification of mu1140-COOH. The red X or black circlesindicate residues with no signal acquired.

FIG. 7. In vitro lipid II binding assay. TLC plate assay of mutacin 1140or mu1140-COOH mixed with lipid II. Binding of lipid II will keep lipidII at the origin. Lipid II or mu1140-COOH by itself was used as anegative control showing no staining. Lipid II and mutacin 1140 was usedas a positive control for trapping lipid II at the origin.

FIG. 8. Overlay assay comparing the zone of inhibitions for selectmutations within the core peptide of mutacin 1140. The bioactivity iscompared to the relative activity (zone of Inhibition) of the wild-typestrain. The deletion strain of mutA is used as a negative control.

FIGS. 9-10. Mature forms of certain lantibiotics. Mutacin B-Ny266 (SEQID NO: 39); Mutacin 1140 (SEQ ID NO: 33); Microbisporicin (SEQ ID NO:40); Mutacin I (SEQ ID NO: 41); Epidermin (SEQ ID NO: 34); Gallidermin(SEQ ID NO: 42); Mersacidin (SEQ ID NO: 43); Lichenicidin A2 (SEQ ID NO:44); Lactocin S (SEQ ID NO: 45); Mutacin II (SEQ ID NO: 46); Epidermin(SEQ ID NO: 34); Gallidermin (SEQ ID NO: 42); Salivaricin (SEQ ID NO:47); Lacticin 3147—A2 (SEQ ID NO: 48); Actagardine (SEQ ID NO: 49);Mersacidin (SEQ ID NO: 43); Mutacin B-hy266 (SEQ ID NO: 39); Mutacin1140 (SEQ ID NO: 33); Ruminococcin A (SEQ ID NO: 50); andMicrobisporicin (SEQ ID NO: 40).

FIG. 11. Bioactivity of core peptide mutants compared to wild-type S.mutans. The bioactivity is represented as the ratio of the zone ofinhibition of mutant to wild-type strain. * denotes statisticalsignificance.

FIGS. 12A-12B. Pharmacokinetic and pharmacodynamic analysis of mutacin1140 and mutacin 1140 core peptide mutants. A. Plasma concentrations ofnative mutacin 1140 (blue), R13A (orange), S5G (yellow), and S5A:T14G(grey) following an intravenous administration of a 2.5 mg/Kg dose. B.Kill kinetics of wild-type mutacin 1140 (blue; at 0.5 μg/ml) and R13A(orange; at 0.125 μg/ml) against S. pneumoniae ATCC 27336.

FIG. 13. Typsin stability of mutacin 1140 core peptide mutants. WTdenotes wild-type mutacin 1140. Overlay assay was done using M. luteusas an indicator strain.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO: 1-13: Sequences of exemplary lantibiotic propeptides.

SEQ ID NO: 14-26: Sequences of exemplary mature lantibiotics.

SEQ ID NO: 27: Forward primer for mutD replacement.

SEQ ID NO: 28: Reverse primer for mutD replacement.

SEQ ID NO: 29: Forward primer for mutD replacement.

SEQ ID NO: 30: Reverse primer for mutD replacement.

SEQ ID NO: 31: Forward primer for mutD clean deletion.

SEQ ID NO: 32: Reverse primer for mutD clean deletion.

DETAILED DISCLOSURE OF THE INVENTION

As used herein, the singular forms “a”, “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise. Further, to the extent that the terms “including”,“includes”, “having”, “has”, “with”, or variants thereof are used ineither the detailed description and/or the claims, such terms areintended to be inclusive in a manner similar to the term “comprising”.The transitional terms/phrases (and any grammatical variations thereof)“comprising”, “comprises”, “comprise”, include the phrases “consistingessentially of”, “consists essentially of”, “consisting”, and“consists”.

The phrases “consisting essentially of” or “consists essentially of”indicate that the claim encompasses embodiments containing the specifiedmaterials or steps and those that do not materially affect the basic andnovel characteristic(s) of the claim.

“Pharmaceutically acceptable carrier” or “pharmaceutically acceptableexcipient” includes any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents and the like. The use of such media and agents forpharmaceutically active substances is well known in the art. Exceptinsofar as any conventional media or agent is incompatible with themodified lantibiotics described herein, its use in the pharmaceuticalcompositions of the invention is contemplated.

“Treatment”, “treating”, “palliating” and “ameliorating” (andgrammatical variants of these terms), as used herein, are usedinterchangeably. These terms refer to an approach for obtainingbeneficial or desired results including but not limited to therapeuticbenefit. A therapeutic benefit is achieved with the eradication oramelioration of one or more of the physiological symptoms associatedwith the underlying disorder such that an improvement is observed in thepatient, notwithstanding that the patient may still be afflicted withthe underlying disorder, for example, a bacterial infection.

The term “effective amount” or “therapeutically effective amount” refersto that amount of an inhibitor described herein that is sufficient toeffect the intended application including but not limited to diseasetreatment, for example, clearing a bacterial infection. Thetherapeutically effective amount may vary depending upon the intendedapplication (in vitro or in vivo), or the subject and disease conditionbeing treated, e.g., the weight and age of the subject, the severity ofthe disease condition, the manner of administration and the like, whichcan readily be determined by one of ordinary skill in the art. Thespecific dose will vary depending on the particular lantibiotic chosen,the dosing regimen to be followed, whether it is administered incombination with other compounds, timing of administration, the tissueto which it is administered, and the physical delivery system in whichit is carried.

“Subject” refers to an animal, such as a mammal, for example a human.The methods described herein can be useful in both humans and non-humananimals. In some embodiments, the subject is a mammal.

As used herein, “a lantibiotic” is a protein produced by a bacteriumthat is active against another bacterium and that contains one or morethioether bridges called lanthionines. A lantibiotics contains one ormore amino acids having PTMs, for example, dehydrations of serine andthreonine residues into dehydroalanine and dehydrobutyrine residues,respectively. In addition, these dehydrated residues are cyclized withcysteines to form thioether bridges called lanthionines.

The epidermin group of lantibiotics and certain other lantibiotics haveS—[(Z)-2-aminovinyl]-D-cysteine (AviCys) residue at the C-terminal endof the lantibiotic (FIG. 1a ). This residue is produced bydecarboxylation of the cysteine at the C-terminus of precursorlantibiotic peptide. AviCys forms a lanthionine ring with otherpost-translationally modified amino acids of the lantibiotic peptide.

As used herein, “a lantibiotic peptide” refers to a peptide that doesnot contain any PTMs. Post-translational modification of the lantibioticpeptide produces a lantibiotic.

As used herein, a “precursor lantibiotic” refers to a peptide thatcontains an intact cysteine at the C-terminus of the lantibiotic, i.e.,a cysteine at the C-terminus of the precursor lantibiotic that is notdecarboxylated and that contains a free carboxyl group. A “precursorlantibiotic” may or may not contain the other PTMs present in thecorresponding lantibiotic.

The term “modified lantibiotic” refers to a lantibiotic that containsall the other PTMs present in a lantibiotic, except the PTMs to thecysteine at the C-terminus of the lantibiotic. Accordingly, in amodified lantibiotic, the cysteine at the C-terminus is notdecarboxylated and contains a free carboxyl group and the modifiedlantibiotic contains all the other PTMs present in the correspondinglantibiotic.

The term “parent lantibiotic” is used herein to distinguish a “modifiedlantibiotic” or a “functionalized lantibiotic” from the corresponding“lantibiotic”. For example, a “modified mutacin” contains all the otherPTMs present in mutacin, except the PTMs to the cysteine at theC-terminus of mutacin. Similarly, a “functionalized mutacin” containsall the other PTMs present in mutacin, except the PTMs to the cysteineat the C-terminus of mutacin and wherein the carboxyl group of thecysteine at the C-terminus is conjugated to a moiety.

A “native gene” or “an endogenous gene” is a gene that is naturallyfound in a bacterium; whereas, an “exogenous gene” is a gene introducedinto a bacterium and which was obtained from an organism other thebacterium.

The invention relates to the importance of the C-terminalS—[(Z)-2-aminovinyl]-D-cysteine (AviCys) residue for antibacterialactivity of lantibiotics. The PTM for making the AviCys residue isessential for the lateral assembly mechanism of activity that trapslipid II into a large complex.

Accordingly, one embodiment of the invention provides a modifiedlantibiotic having an intact cysteine at the C-terminus. Non-limitingexamples of parent lantibiotics that correspond to the modifiedlantibiotics described herein include lichenicidin (e.g., SEQ ID NO: 1),lactocin-S (e.g., SEQ ID NO: 2), salivaricin (e.g., SEQ ID NO: 3),mutacin (e.g., SEQ ID NOs: 4, 10 and 11), lacticin (e.g., SEQ ID NO: 5),actagardine (e.g., SEQ ID NO: 6), mersacidin (e.g., SEQ ID NO: 7),epidermin (e.g., SEQ ID NO: 8), gallidermin (e.g., SEQ ID NO: 9),ruminococcin (e.g., SEQ ID NO: 12) or microbisporicin (SEQ ID NO: 13).

The modified lantibiotics having an intact cysteine at the C-terminuscan be further conjugated to moieties through the free carboxyl group ofthe cysteine at the C-terminus. Accordingly, functionalized lantibioticis provided, wherein the carboxyl group of the cysteine at theC-terminus is conjugated to a moiety.

The moiety can be a functional group or a detectable label. Non-limitingexamples of the functional groups that can be conjugated to the carboxylgroup of the cysteine at the C-terminus of the modified lantibioticinclude substituted or unsubstituted chemical groups, such as alkane,alkene, alkyne, haloalkyl, alcohol, ether, amine, aldehyde, ketone, acylhalide, carboxylate, ester, amide, aryl or heteroaryl. Specificembodiments within the genus of chemical groups recited herein are wellknown in the art and such embodiments are within the purview of theinvention.

The carboxyl group of the cysteine at the C-terminus of the modifiedlantibiotic can be covalently joined to a carbon or a heteroatom of thefunctional group.

In one embodiment, the carboxyl group of the cysteine at the C-terminusof the modified lantibiotic is covalently joined to a carbon or thenitrogen atom of an amine. The amine can be a primary or secondaryamine. Non-limiting examples of amines include substituted orunsubstituted forms of alkyl-amines, for example, methylamine ordiaminoheptane, or substituted or unsubstituted forms of aryl-amines orheteroaryl amines, for example, chlorophenylalanine ordi-chlorophenylalanine.

In specific embodiments, the functional group is substituted halo-aryl,for example, chlorophenylalanine or di-chlorophenylalanine.

In another embodiment of the functionalized lantibiotic, the moiety is adetectable label. The detectable label can be a fluorescent label,radiolabel or bioluminescent label.

Numerous commercially available fluorescent labels are suitable forconjugation to the modified lantibiotic described herein, for example,fluorescein, dR110, 5-FAM™ 6-FAM™, dR6G, JOE™, HEX™, VIC®, TET™,dTAMRA™, TAMRA™, NED™, dROX™, ROX™, PET® and LIZ®. Additional examplesof fluorescent labels suitable for the invention described herein areknown to a skilled artisan and such embodiments are within the purviewof the invention.

Non-limiting examples of parent lantibiotics that correspond to thefunctionalized lantibiotics described herein include lichenicidin (e.g.,SEQ ID NO: 1), lactocin-S (e.g., SEQ ID NO: 2), salivaricin (e.g., SEQID NO: 3), mutacin (e.g., SEQ ID NOs: 4, 10 (mutacin ny266) and 11(mutacin 1140/mutacin III)), lacticin (e.g., SEQ ID NO: 5), actagardine(e.g., SEQ ID NO: 6), mersacidin (e.g., SEQ ID NO: 7), epidermin (e.g.,SEQ ID NO: 8), gallidermin (e.g., SEQ ID NO: 9), ruminococcin (e.g., SEQID NO: 12) or microbisporicin (SEQ ID NO: 13).

In certain embodiments, a parent lantibiotic that corresponds to thefunctionalized lantibiotics described herein comprises a sequence thatis homologous to the sequence of a known lantibiotic, for example,lichenicidin (e.g., SEQ ID NO: 1), lactocin-S (e.g., SEQ ID NO: 2),salivaricin (e.g., SEQ ID NO: 3), mutacin (e.g., SEQ ID NOs: 4, 10 and11), lacticin (e.g., SEQ ID NO: 5), actagardine (e.g., SEQ ID NO: 6),mersacidin (e.g., SEQ ID NO: 7), epidermin (e.g., SEQ ID NO: 8),gallidermin (e.g., SEQ ID NO: 9), ruminococcin (e.g., SEQ ID NO: 12) ormicrobisporicin (SEQ ID NO: 13). A lantibiotic that is homologous toknown lantibiotic shares at least about 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% sequence identity with the sequences of thecorresponding known lantibiotic, for example, lichenicidin (e.g., SEQ IDNO: 1), lactocin-S (e.g., SEQ ID NO: 2), salivaricin (e.g., SEQ ID NO:3), mutacin (e.g., SEQ ID NOs: 4, 10 and 11), lacticin (e.g., SEQ ID NO:5), actagardine (e.g., SEQ ID NO: 6), mersacidin (e.g., SEQ ID NO: 7),epidermin (e.g., SEQ ID NO: 8), gallidermin (e.g., SEQ ID NO: 9),ruminococcin (e.g., SEQ ID NO: 12) or microbisporicin (SEQ ID NO: 13).

For the purpose of this invention, when a sequence of lantibiotic isrepresented by a sequence identifier, the sequence in the sequenceidentifier provides the amino acids sequence of the lantibiotic prior toany PTMs. The amino acids provided in a sequence that corresponds to alantibiotic are post-translationally modified to produce a lantibiotichaving antibacterial activity. Such PTMs include, for example,dehydrations of serine and threonine residues into dehydroalanine anddehydrobutyrine residues, respectively; cyclization of the dehydratedresidues with cysteines to form thioether bridges called lanthionines;formation of D-alanines (e.g., in lacticin 3147); formation of β-hydroxyaspartate (e.g., in cinnamycin); formation of 2-oxopropionyl (e.g., inlactocin S); and oxidation of lanthionine (e.g., in actagardine);formation of AviCys at the C-terminal end of the peptide (e.g., inmutacin).

A person of ordinary skill in the art can design homologs of a givenlantibiotic by substituting the amino acids within a lantibioticsequence that are not likely to affect the activity of the lantibiotichaving an amino acid substitution. For example, a person of ordinaryskill in the art can substitute amino acids that are not modified viaPTMs in a lantibiotic, particularly, with other amino acids that havesimilar chemical properties via conservative amino acid substitutions.

Conservative amino acid substitutions are changes in a protein sequencethat change an amino acid to a different amino acid with similarbiochemical properties, e.g. charge, hydrophobicity and size. Certainexamples of conservative substitutions are provided in Table 1 below:

TABLE 1 Conservative amino acid substitutions to produce homologs of alantibiotic Original residue Substitution Ala (A) Val (V), Leu (L), Ile(I) Arg (R) Lys (K), Gln (Q), Asn (N) Asn (N) Gln (Q), His (H), Lys (K),Arg (R) Asp (D) Glu (E) Cys (C) Ser (S) Gln (Q) Asn (N) Glu (E) Asp (D)His (H) Asn (N), Gln (Q), Lys (K), Arg (R) Ile (I) Leu (L), Val (V), Met(M), Ala (A), Phe (F) Leu (L) Ile (I), Val (V), Met (M), Ala (A), Phe(F) Lys (K) Arg (R), Gln (Q), Asn (N) Met (M) Leu (L), Phe (F), Ile (I)Phe (F) Leu (L), Val (V), Ile (I), Ala (A) Pro (P) Gly (G) Ser (S) Thr(T) Thr (T) Ser (S) Trp (W) Tyr (Y) Tyr (Y) Trp (W), Phe (F), Thr (T),Ser (S) Val (V) Ile (I), Leu (L), Met (M), Phe (F), Ala (A).

In certain embodiments, the invention provides mutant lantibioticshaving one or more mutations in the core peptide. Such mutants arecalled core peptide mutants. Certain of the core peptide mutants of theinvention exhibit higher activity, improved pharmacokinetics, higherstability, or a combination thereof, compared to the correspondingunmodified lantibiotic.

In specific embodiments, the invention provides core peptide mutants ofmutacin, particularly, mutacin 1140 comprising the sequence of SEQ IDNO: 24 or mutacin B-Ny266 comprising the sequence of SEQ ID NO: 23. Inother embodiments, a core peptide mutant of mutacin 1140 or mutacinB-Ny266 comprises or consists of a mutation provided in the Table 2below.

TABLE 2 Examples of core peptide mutants of mutacin 1140 comprising SEQID NO: 24 or mutacin B-Ny266 comprising the sequence of SEQ ID NO: 23containing a single amino acid mutation/substitution. For mutacin 1140,the amino acid positions are indicated with respect to SEQ ID NO: 24 andfor mutacin B-Ny266, the amino acid positions are indicated with respectto SEQ ID NO: 23. Mutations in the corresponding positions of mutacin1140 or mutacin B-Ny266 prior to post-translational modificationcomprising SEQ ID NO: 10 or 11, respectively, are also envisioned. Aminoacid position Original amino acid Replacement amino acid 5 SerineGlycine 5 Serine Threonine 5 Serine Glutamate 5 Serine Alanine 13Arginine Alanine 14 Threonine Glycine 14 Threonine Alanine 15 GlycineAlanine 12 Alanine Threonine 4 Tryptophan Serine 6 Leucine Serine

In other embodiments, a core peptide mutant of mutacin 1140 comprises orconsists of a combination of mutations described in the Table 3 below.

TABLE 3 Examples of core peptide mutants of mutacin 1140 comprising SEQID NO: 24 or mutacin B-Ny266 comprising the sequence of SEQ ID NO: 23.For mutacin 1140, the amino acid positions are indicated with respect toSEQ ID NO: 24 and for mutacin B-Ny266, the amino acid positions areindicated with respect to SEQ ID NO: 23. Mutations in the correspondingpositions of mutacin 1140 or mutacin B-Ny266 prior to post-translationalmodification comprising SEQ ID NO: 10 or 11, respectively, are alsoenvisioned. Amino acid Original amino acids Replacement amino acidpositions (respectively) (respectively) 12, 14  Alanine and ThreonineThreonine and Glycine 13, 14  Arginine and Threonine Alanine and Alanine14, 15  Threonine and Glycine Alanine and Alanine 5, 14 Serine andThreonine Glycine and Glycine 5, 14 Serine and Threonine Alanine andGlycine 5, 14 Serine and Threonine Threonine and Glycine 5, 14 Serineand Threonine Alanine and Serine 5, 14 Serine and Threonine Alanine andAlanine 5, 14 Serine and Threonine Glycine and Alanine 5, 14 Serine andThreonine Glutamate and Alanine 5, 14 Serine and Threonine Threonine andAlanine 5, 12 Serine and Alanine Alanine and Serine 5, 13 Serine andArginine Alanine and Serine 13, 14 and 15 Arginine, Threonine andAlanine, Alanine and Glycine Alanine  5, 13 and 14 Serine, Arginine andGlcyine, Alanine and Threonine Alanine  4, 5 and 14 Tryptophan, Serineand Serine, Alanine and Threonine Alanine  5, 6 and 14 Serine, Leucineand Alanine, Serine and Threonine Alanine  5, 12 and 14 Serine, Alanineand Alanine, Serine and Threonine Alanine  5, 13 and 14 Serine, Arginineand Alanine, Serine and Threonine Alanine 12, 13 and 14 Alanine,Arginine and Glycine, Glycine and Threonine Glycine

In further embodiments, the invention provides a core peptide mutant ofmutacin 1140 comprising SEQ ID NO: 24 or mutacin B-Ny266 comprising thesequence of SEQ ID NO: 23, with the proviso that the core peptide mutantdoes not contain:

i) amino acid mutations of one or any combination of the second, sixthor thirteenth amino acid residue of SEQ ID NO: 23 or 24 (i.e., thesecond alone, the sixth alone, the thirteenth alone, the combination ofthe second and sixth, the combination of the second and thirteenth, thecombination of the sixth and thirteenth, or the combination of thesecond, sixth and thirteenth amino acid residues of SEQ ID NO: 23 or24);

ii) mutation of one or more of Leu6 and Arg13 to an amino acid selectedfrom alanine, glycine, valine, leucine or isoleucine;

iii) mutation of one or more of Leu6 and Arg13 to alanine; or

iv) mutation of Lys2 to an amino acid selected from alanine, glycine,valine, leucine or isoleucine.

A further embodiment of the invention provides a pharmaceuticalcomposition comprising a modified lantibiotic or a functionalizedlantibiotic described herein and a pharmaceutically acceptable carrieror excipient. Further embodiments of the invention provide a method oftreating a bacterial infection in a subject by administering to thesubject a modified lantibiotic or a functionalized lantibiotic describedherein. The modified lantibiotic or a functionalized lantibioticadministered to a subject can be in the form of pharmaceuticalcompositions of the invention.

Decarboxylation of the terminal cysteine in a lantibiotic is typicallyperformed by a decarboxylase, which is typically a flavoprotein. Thisdecarboxylase is specific for C-terminal cysteines from a lantibioticpeptide. Decarboxylation of the C-terminal cysteine produces anene-thiol intermediate that promotes terminal ring formation.Accordingly, a further embodiment of the invention provides a bacteriumthat synthesizes a modified lantibiotic. Such bacterium is produced by agenetic modification to a wild-type or a parent bacterium thatsynthesizes a parent lantibiotic that corresponds to the modifiedlantibiotic.

Accordingly, certain embodiments of the invention provide a bacteriumthat synthesizes a modified lantibiotic, wherein the modifiedlantibiotic has an intact cysteine at the C-terminus, and wherein thebacterium is genetically modified to inactivate a gene that encodes adecarboxylase enzyme that decarboxylates the cysteine at the C-terminusof a precursor lantibiotic.

Several bacteria are known to produce a lantibiotic, for example,Bacillus licheniformis (strain ATCC 14580), Streptomyces sp.,Lactobacillus sakei, Streptococcus salivarius, Streptococcus mutans,Lactococcus lactis, Actinoplanes liguriensis, Bacillus sp.,Staphylococcus epidermidis, Staphylococcus gallinarum, Streptococcusmutans or Ruminococcus gnavus. Accordingly, certain embodiments of theinvention provide a bacterium that synthesizes a modified lantibiotic,wherein the bacterium is Bacillus licheniformis (strain ATCC 14580),Streptomyces sp., Lactobacillus sakei, Streptococcus salivarius,Streptococcus mutans, Lactococcus lactis, Actinoplanes liguriensis,Bacillus sp., Staphylococcus epidermidis, Staphylococcus gallinarum,Streptococcus mutans or Ruminococcus gnavus.

Non-limiting examples of bacterial strains, corresponding lantibioticsand their peptide sequences before the PTMs are provided in Table 4below.

TABLE 4 Examples of organisms producing lantibiotic and the sequences ofunmodified peptides corresponding to mature forms of lantibiotics.Sequences in mature Sequences Name of the form of propeptide Name of theorganism lantibiotic (SEQ ID) (SEQ ID) Bacillus licheniformislichenicidin A2 1 14 (strain ATCC 14580) Lactobacillus sakei L45lactocin-S 2 15 Streptococcus salivarius Salivaricin 3 16 Streptococcusmutans Mutacin-2 4 17 Lactococcus lactis lacticin 3147 5 18 subsp.lactis A2 (Streptococcus lactis) Actinoplanes liguriensis actagardine 619 Bacillus sp. mersacidin 7 20 (strain HIL-Y85/54728) Staphylococcusepidermidis Epidermin 8 21 Staphylococcus gallinarum Gallidermin 9 22Streptococcus mutans mutacin B-Ny266 10 23 Streptococcus mutansmutacin-1140 11 24 Ruminococcus gnavus Ruminococcin-A 12 25 Microbisporacorallina Microbisporicin 13 26

For a given bacterium that produces a lantibiotic, a person of ordinaryskill in the art can determine which gene encodes a decarboxylase enzymethat decarboxylates the cysteine at the C-terminus of a precursorlantibiotic. For example, EpiD gene from S. epidermidis encodes for adecarboxylase enzyme that decarboxylates the cysteine at the C-terminusof a precursor epidermin. Similarly, MrsD gene from Bacullus sp. encodesfor a decarboxylase enzyme that decarboxylates the cysteine at theC-terminus of a precursor mersacidin. Additional examples ofdecarboxylase enzymes that decarboxylate the cysteine at the C-terminusof a precursor lantibiotic in other lantibiotic producing bacteria areknown in the art and such embodiments are within the purview of theinvention.

A gene that encodes the decarboxylase enzyme can be inactivated in anumber of genetic modification techniques. For example, a gene thatencodes the decarboxylase enzyme can be inactivated by deletion,frameshift mutation(s), point mutation(s), antisense RNA, the insertionof stop codon(s), or combinations thereof. For example, target genes canbe inactivated by the introduction of insertions, deletions, or randommutations into the gene that encodes the decarboxylase enzyme. Thus,certain embodiments of the invention provide for the insertion of atleast one stop codon (e.g., one to ten or more stop codons) into thegene that encodes the decarboxylase enzyme. Some embodiments of theinvention provide for the insertion or deletion of 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29 or 30 or more bases in order to introduce a frameshiftmutation in a gene that encodes the decarboxylase enzyme gene. Otherembodiments of the invention provide for the insertion or deletion of 1,2, 4, 5, 7, 8, 10, 11, 13, 14, 16, 17, 19, 20, 22, 23, 25, 26, 28, 29 ormore bases in order to introduce a frameshift mutation in a gene thatencodes the decarboxylase enzyme. Yet other embodiments of the subjectapplication provide for the introduction of one or more point mutations(e.g., 1 to 30 or more) within a gene that encodes the decarboxylaseenzyme while other embodiments of the invention provide for the partial,total or complete deletion of a gene that encodes the decarboxylaseenzyme.

In one embodiment, a gene that encodes the decarboxylase enzyme thatdecarboxylates the cysteine at the C-terminus of a precursor lantibioticin a bacterium is inactivated by introducing into the bacterium aninhibitory RNA specifically directed to the gene. In certainembodiments, the inhibitory RNA is an antisense RNA that has anappropriate sequence to inhibit the expression of an mRNA that encodesthe decarboxylase enzyme.

Various techniques for carrying out the genetic modifications toinactivate a gene that encodes the decarboxylase enzyme in a bacteriumare well known in the art and such embodiments are within the purview ofthe invention.

In one embodiment, a bacterium that produces a lantibiotic containsendogenous gene cluster that encodes the lantibiotic, i.e., thebacterium naturally produces the lantibiotic. Such bacterium can begenetically modified to inactivate the gene that encodes thedecarboxylase enzyme that decarboxylates the cysteine at the C-terminusof a precursor lantibiotic.

In another embodiment, a bacterium that produces a lantibiotic containsexogenous gene cluster that encodes the lantibiotic, i.e., the bacteriumis genetically engineered to express the gene cluster that encodes thelantibiotic. Such bacterium can be further genetically modified toinactivate the gene that encodes the decarboxylase enzyme thatdecarboxylates the cysteine at the C-terminus of a precursorlantibiotic.

In a further embodiment, a bacterium that produces a lantibiotic throughexogenous genes that encode the lantibiotic, i.e., the bacterium isgenetically engineered to express the genes that encode the lantibioticdoes not contain an endogenous gene that encodes the decarboxylaseenzyme that decarboxylates the cysteine at the C-terminus of a precursorlantibiotic. In such bacterium, genetic modification to inactivate thegene that encodes the decarboxylase enzyme that decarboxylates thecysteine at the C-terminus of a precursor lantibiotic is not necessary.As such, an embodiment of the invention provides a bacterium that doesnot contain an endogenous gene that encodes the decarboxylase enzymethat decarboxylates the cysteine at the C-terminus of a precursorlantibiotic and that contains exogenous genes that encode thelantibiotic, i.e., the bacterium is genetically engineered to expressthe genes that encode the lantibiotic.

A further embodiment of the invention provides a method of producing amodified lantibiotic, wherein the modified lantibiotic has an intactcysteine at the C-terminus. The method comprises the steps of:

a) culturing a bacterium that synthesizes the modified lantibiotic,wherein the bacterium is genetically modified to inactivate a gene thatencodes a decarboxylase enzyme that decarboxylates the cysteine at theC-terminus of a precursor lantibiotic,

b) purifying the modified lantibiotic from the culture.

The embodiments of the invention discussed above with respect to abacterium that produces a modified lantibiotic as described herein areapplicable to the methods of producing the modified lantibiotic as well.

In certain embodiments, the bacterium is cultured under appropriateconditions for appropriate period of time. A skilled artisan iswell-versed with the methods of culturing bacteria and such embodimentsare within the purview of the invention.

A step of producing a modified lantibiotic comprises purifying themodified lantibiotic from the culture of the bacterium. Non-limitingexamples of such purification methods include liquid chromatography,particularly, fast protein liquid chromatography (FPLC),high-performance liquid chromatography (HPLC), ion exchangechromatography, size-exclusion chromatography, or affinitychromatography. Additional examples of purifying a modified lantibioticfrom bacterial culture are known to a skilled artisan and suchembodiments are within the purview of the invention.

A further embodiment of the invention provides a method of producing afunctionalized lantibiotic by reacting, under appropriate conditions, amodified lantibiotic described herein with a moiety to conjugate themoiety to the carboxyl group of the terminal cysteine. Various moietiesdescribed above in connection with the functionalized lantibiotic arealso applicable to the methods of making the functionalized lantibioticsdescribed herein.

In one embodiment, a functionalized lantibiotic is produced by1-Hydroxy-7-azabenzotriazole/1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide(HOAt/EDC) coupling method. Additional techniques for conjugating amodified lantibiotic described herein with a moiety are known to askilled artisan and such embodiments are within the purview of theinvention.

An embodiment of the invention provides a lantibiotic containing one ormore amino acid mutations, wherein the lantibiotic containing the one ormore amino acid mutations exhibits higher anti-bacterial activitycompared to the activity of the lantibiotic being mutagenized. In oneembodiment, the invention provides a mutant of a lantibiotic belongingto the epidermin group of lantibiotics, for example, mutacin (e.g., SEQID NOs: 4, 10 and 11), epidermin (e.g., SEQ ID NO: 9), gallidermin(e.g., SEQ ID NO: 10), that contains one or more amino acid mutations,wherein the mutant lantibiotic exhibits higher anti-bacterial activitycompared to lantibiotic being mutagenized.

Non-limiting examples of a mutant of mutacin 1140 that exhibit higherantibacterial activity compared to mutacin 1140 includes mutations toone or more of Leu6 and Arg13. In one embodiment, a mutant mutacin 1140contains mutations of one or more of Leu6 and Arg13 to an amino acidselected from alanine, glycine, valine, leucine or isoleucine. Incertain embodiment, a mutant mutacin 1140 contains mutations of one ormore of Leu6 and Arg13 to alanine.

Non-limiting examples of a mutant of epidermin that exhibit higherantibacterial activity compared to epidermin includes mutations to oneor more of Ile6 and Lys13. In one embodiment, a mutant epidermincontains mutations of one or more of Ile6 and Lys13 to an amino acidselected from alanine, glycine, valine, leucine or isoleucine. Incertain embodiment, a mutant epidermin contains mutations of one or moreof Ile6 and Lys13 to alanine.

Non-limiting examples of a mutant of gallidermin that exhibit higherantibacterial activity compared to gallidermin include mutations to oneor more of Lue6 and Lys13. In one embodiment, a mutant gallidermincontains mutations of one or more of Leu6 and Lys13 to an amino acidselected from alanine, glycine, valine, leucine or isoleucine. Incertain embodiment, a mutant gallidermin contains mutations of one ormore of Leu6 and Lys13 to alanine.

Non-limiting examples of a mutant of mutacin B-Ny266 that exhibit higherantibacterial activity compared to mutacin B-Ny266 includes mutations toone or more of Phe6 and Lys13. In one embodiment, a mutant mutacinB-Ny266 contains mutations of one or more of Phe6 and Lys13 to an aminoacid selected from alanine, glycine, valine, leucine or isoleucine. Incertain embodiment, a mutant mutacin B-Ny266 contains mutations of oneor more of Phe6 and Lys13 to alanine.

A further embodiment of the invention provides a lantibiotic containingone or more amino acid mutations, wherein the lantibiotic containing theone or more amino acid mutations is expressed in higher amounts by thebacterium producing the mutant lantibiotic compared to the bacteriumproducing the lantibiotic being mutagenized.

In one embodiment, the invention provides a mutant of a lantibioticbelonging to the epidermin group of lantibiotics, for example, mutacin(e.g., SEQ ID NOs: 4, 10 and 11), epidermin (e.g., SEQ ID NO: 9),gallidermin (e.g., SEQ ID NO: 10), that contains one or more amino acidmutations, wherein the mutant lantibiotic is expressed in higher amountsby the bacterium producing the mutant lantibiotic compared to thebacterium producing the lantibiotic being mutagenized.

In one embodiment, the invention provides a bacterium producing a mutantof mutacin 1140 (SEQ ID NO: 11) containing one or more amino acidmutations, wherein the mutant of mutacin 1140 is expressed in higheramounts by the bacterium producing the mutant mutacin 1140 compared tothe bacterium producing mutacin 1140.

Non-limiting examples of a mutant mutacin 1140 that are expressed inhigher amounts include a mutation of Lys2. In one embodiment, a mutantmutacin 1140 contains mutations of Lys2 to an amino acid selected fromalanine, glycine, valine, leucine or isoleucine. In certain embodiment,a mutant mutacin 1140 contains a mutation of Lys2 to alanine.

Non-limiting examples of a mutant mutacin B-Ny266 that are expressed inhigher amounts include a mutation of Lys2. In one embodiment, a mutantmutacin B-Ny266 contains mutations of Lys2 to an amino acid selectedfrom alanine, glycine, valine, leucine or isoleucine. In certainembodiment, a mutant mutacin B-Ny266 contains a mutation of Lys2 toalanine.

Certain embodiment of the invention provides a pharmaceuticalcomposition comprising a mutant lantibiotic described herein and apharmaceutically acceptable carrier or excipient. Further embodiments ofthe invention provide a method of treating a bacterial infection in asubject by administering to the subject a mutant lantibiotic describedherein. The mutant lantibiotic administered to a subject can be in theform of pharmaceutical compositions of the invention.

Further embodiments of the invention provide a bacterium expressing amutant lantibiotic, particularly, a mutant mutacin 1140 that exhibitshigher activity compared to mutacin 1140 or a mutant mutacin 1140 thatexhibits higher expression by a bacterium compared to mutacin 1140.Various embodiments discussed above with respect to mutants of mutacin1140 are also applicable to a bacterium expressing a mutant mutacin1140. A skilled artisan can readily design a bacterium expressing amutant mutacin 1140 by transforming a bacterium with a gene or a genecluster encoding a mutant mutacin 1140 and such embodiments are withinthe purview of the invention.

Materials and Methods

Bacterial Strains and Media

The bacterial strains and plasmids used in this study are outlined inTable 5. Streptococcus mutans strains, Bacillus subtilis PY79,Streptococcus pneumoniae, and Micrococcus luteus ATCC 10240 were grownin either THyex media agar (30 g/L Todd Hewitt Broth, 3 g/L yeastextract, 15 g/L agar; Bacto, Sparks, Md.), THyex broth (30 g/L ToddHewitt Broth, 3 g/L yeast extract), or THyex top agar (30 g/L ToddHewitt Broth, 3 g/L yeast extract, 7.5 g/L agar; Bacto, Sparks, Md.). E.coli was grown in LB medium (10 g/L tryptone, 5 g/L yeast extract, and10 g/L NaCl, pH adjusted with NaOH to pH 7.5), Terrific Broth (12 g/Ltryptone, 24 g/L yeast extract, 4 mL/L glycerol, and 2.2 g/L KH₂PO₄ and9.4 g/LK₂HPO₄), or LB plates (10 g/L tryptone, 5 g/L yeast extract, and10 g/L NaCl, 15 g/L agar, and pH adjusted with NaOH to pH 7.5).

TABLE 5 Strains used in this study. Reference Plasmid Relevant or Strainintermediate characteristic Source S. mutans JH1140 WT bacteriocinStrain 1, (ATCC 55676) producing strain ATCC S. mutans ΔmutA mutAdeletion 2 strain S. mutans IFDC2:mutD mutD gene This study replacementwith IFDC2 cassette S. mutans ΔmutD pΔmutD Clean deletion This study ofmutD M. luteus ATCC 10240 Indicator strain 3 for activity B. subtilisPY79 Indicator strain 4 for activity S. pneumoniae ATCC Indicator strainATCC 27336 for activity E. coli DH5α pCR2.1 and Intermediate InvitrogenpET28B(+) cloning host E. coli BL21 Protein Invitrogen overexpressionhost E. coli pmutD-kan pmutD-kan Codon optimized This study mutD E. colipet28B:mutD pet28B:mutD MutD This study overexpression strain

Gene Deletion of MutD

Primers used for both sequencing and mutagenesis were designed using theS. mutans genome and lan cluster (GenBank/EMBL accession numberAF051560) (Table 6). The IFDC2 gene replacement system was used, aspreviously described, to mutate or delete MutD³⁰. In-frame deletioncassette (IFDC2) is a gene replacement cassette containing both apositive selection marker (ermAM) and a negative selection marker(-pheS*). Approximately 500 bp upstream of mutD was amplified and toprevent polar effects of downstream genes of mutD, a 500 bp fragment wasamplified starting at 100 bp upstream of the stop codon and 400 bpdownstream of mutD. The fragments were attached to the 5′ and 3′ end ofthe IFDC2 cassette, respectively, by PCR. The final 3 kb fragment wastransformed into S. mutans ATCC 55676 by competent stimulating peptide(CSP) protocol⁵⁷. An overnight culture of S. mutans ATCC 55676 wasdiluted to 0.1 OD⁶⁰⁰ and incubated at 37° C. to 0.25 OD⁶⁰⁰. Two μl of 10μg/mL solution of CSP was then added to 200 μl of the 0.25 OD culture.After a 30 minute incubation time at 37° C., 1 μl of the PCR amplifiedproduct was added to the culture. The transformation was incubated at37° C. for 5 hours before plating 50 μl of a 1000-fold dilution onto aTHyex plate containing 15 μg/mL of erythromycin. Transformants wereconfirmed by PCR. The PCR products were inserted into a Topo PCR2.1®plasmid and were sent for sequencing. Both upstream and downstreamregions were joined together and amplified to create the ΔmutD fragment.The ΔmutD fragment was transformed into S. mutans IFDC2:mutD, andselected on THyex plates (containing 4 mg/mL of P-Chloro-phenylalanine).

TABLE 6 Primers used in this study. Character- Primer Sequence (5′to 3′) istic MutD- GAT TTG TTT CGT AAA GAG GGT TC mutD gene UpF(SEQ ID NO: 27) replacement MutD- CTA CAT CAA TCC CAG AAT CAA CmutD gene DnR (SEQ ID NO: 28) replacement MutD-GAGTGTTATTGTTGCTCGGAAATTATTTCTC mutD gene UpR-IDHCGTTCAG TTAA (SEQ ID NO: 29) replacement MutD-GGTATACTACTGACAGCTTCGGTAATTGTT mutD gene DnF-ermGGACAAGAATC (SEQ ID NO: 30) replacement DelMutD-TTAACTGAACGGAGAAATAATTGGTAATTG mutD clean FTTGGACAAGAATC (SEQ ID NO: 31) deletion DelMutD-GATTCTTGTCCAACAATTACCAATTATTTCT mutD clean RCC GTTCAGTTAA (SEQ ID NO: 32) deletion

Bioactivity Assays

The deferred antagonism assay was performed as previously described³⁹ .S. mutans strains were grown overnight in THyex broth at 37° C. Thecultures were diluted to 0.1 OD OD₆₀₀ and grown to mid-logarithmic phasebefore diluting to 0.05 OD₆₀₀. Then, 2 μl of the cultures were spottedon THyex plates in duplicates of triplicates. The wild-type S. mutansJH1140 and S. mutans ΔmutA were used as positive and negative controlsfor activity, respectively. The plates were incubated for 18 hours in acandle jar at 37° C. After incubation, the strains were heat killed at65° C. for 90 minutes. Fresh M. luteus grown overnight at 37° C. onTHyex plates were used to inoculate pre-warmed THyex broth. The culturewas grown to 0.6 to 0.8 OD before diluting to 0.2 OD₆₀₀. The culture wasfurther diluted 25-fold in melted (42° C.) THyex top agar. Approximately5 mL of the top agar solution was spread on the heat killed bioassayplates and allowed to cool for 10 minutes. The plates were then placedin the incubator (at 37° C.) for 18 hours.

Minimum Inhibitory Concentrations (MICs) were determined according topreviously published protocol³¹. A stock solution of the antibioticstested was first suspended in 50% acetonitrile (ACN) at a concentration640 μg/mL. This stock was subsequently diluted 2-fold until a finalconcentration 0.156 μg/mL was reached. Subsequently, 10 μl of eachdilution was placed into a well on a 96 well microtiter plate. M. luteusATCC 10240, S. pneumoniae, and B. subtilis PY79 were grown overnight inTHyex at 37° C. Cultures were diluted in the morning to 0.1 OD₆₀₀ andallowed to grow to 0.6 OD₆₀₀. This culture was diluted a 100-fold infresh THyex media and then 400 μl of this culture was added to 10 mL offresh THyex. The suspension contains approximately 10⁵ colony formingunits (CFUs). The bacterial suspension (190 μL) was added to each wellcontaining 10 μL of antibiotic suspension or solvent blank. Thisresulted in another 20-fold dilution of the antibiotic suspension. Forthe competition assays, 10⁵ CFU bacterial suspension was initially mixedwith mu1140-COOH for 15 minutes at the 1×MIC of either mutacin 1140(0.25 μg/mL and 0.125 μg/mL for B. subtilis and M. luteus, respectively)or nisin (0.5 μg/mL for B. subtilis and M. luteus). Following the 15minute pretreatment, 190 μL was added to each well as described above.The MIC is described as the highest concentration of antibiotic thatprevented visible growth after 24 hours.

Production and Purification of Mutacin 1140 and mu1140-COOH

Lanthipeptides isolated in this study were cultured as statedpreviously⁵⁸ . S. mutans strains were grown in a modified THyex media.The media contained 30 g/L Todd Hewitt, 3 g/L yeast extract, 1 g/LNaH₂PO₄, 0.2 g/L Na₂HPO₄, 0.7 g/L MgSO₄, 0.005 g/L FeSO₄, 0.005 g/LMnSO₄, and 0.3% agar. The semi-solid agar (1 L) was inoculated withvarious strains of S. mutans and incubated at 37° C. for 72 hours. Afterincubation, the inoculum was frozen at −80° C. overnight and thawed for1 hour in a 65° C. water bath. The inoculum was then centrifuged at20,000 g for 30 minutes and the supernatant was collected. Thesupernatant was mixed with chloroform at 1:1 ratio and mixed vigorously.This mixture was again centrifuged at 20,000 g for 30 minutes. Theprecipitate between both the aqueous and chloroform phases was collectedand dried overnight. The dried product was resuspended in 35% ACNcontaining 0.1% trifluoroacetic acid (TFA) and ran on either a semi-prepC18 column (Agilent® ZORBAX, ODS, C18, 5 μm, 4.6×250 mm) or analyticalcolumn. Peaks collected were confirmed by mass on a Shimadzu® MALDI-MSon both linear and reflectron modes.

Chemical Modification of mu1140-COOH and Nisin

Labeling of C-terminal carboxyl group with methylamine (33% in EtOH)(Sigma-Aldrich), diaminoheptane (Sigma-Aldrich), chlorophenylalanine(Sigma-Aldrich), di-chlorophenylalanine (Sigma-Aldrich), or5-(aminoacetamido)fluorescein (Sigma-Aldrich) was done by1-Hydroxy-7-azabenzotriazole/1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide(HOAt/EDC) coupling. The labeling was done in according to a previouslydescribed method^(16, 39). The reaction mixture was suspended in 100 μlof Dimethyl Formamide (DMF) with 50 nmols of either nisin, orMu1140-COOH, 50 nmols AAA-fluorescein or 200 nmols of the primary amine,60 nmols of 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), and 60nmols of 1-Hydroxy-7-azabenzotriazole (HOAt). The reaction was coveredin foil and incubated at room temperature for approximately 16 hours.The reaction was subsequently diluted 10-fold with 35% ACN containing0.1% TFA and ran on an analytical C-18 column. The labeled peptides wereconfirmed by MALDI-MS as described above. The amount of chemicallyconjugated product was determined by Bradford Assay using manufacturerrecommended protocol (Sigma-Aldrich). The purified labeled peptides werethen dried down and resuspended in 35% ACN containing 0.1% TFA at aconcentration of 100 ug/mL and stored at −20° C.

Microscopy

B. subtilis PY79 was grown and treated with fluorescein labeled peptideas previously described³⁹. A fresh plate of B. subtilis was used toinoculate THyex broth and incubated approximately 16 hours at 37° C. Theculture was diluted 20-fold and placed back in the incubator for 3 hoursat 37° C. Then, 100 μl of the culture was incubated with the antibiotic(10 μg/mL) for 15 minutes. The cells were pelleted and resuspended in100 μl of phosphate buffer solution (PBS). The wash step was repeatedthree times before fixing with 1.6% formaldehyde in PBS. After fixationthe cells were washed with PBS three more times and the remaining pelletwas suspended in 50 μL of PBS. The sample (30 μL) was added to a slideand observed using an Olympus confocal microscope with a 100×/0.90 dryobjective. A 488 nm argon laser was used to excite the fluorophore. Forthe competition assay, the bacterial culture was initially incubatedwith mu1140-COOH or native mutacin 1140 at a concentration of 10 μg/mLfor 15 minutes, washed, and resuspended in fresh media. 10 μg/mL offluorescein labeled nisin was then added for 15 minutes, beforefollowing the wash protocol previously stated.

Mu1140 Double Labeling and Edman Sequencing

Edman sequencing has been frequently used to determine the sequence ofsmall peptides, such as lantibiotics⁵⁹⁻⁶⁰. Mu1140-COOH was doublylabeled as previously described for mutacin 1140³². A 200 μM solution ofMu1140-COOH in 5 μl of water was added to a reaction tube containing 2mg of sodium borohydride. Then, 94 μL of solution B (570 mg guanidineHCl, 100 mL N-ethylmorpholine and water to a final volume of 1 mL; thepH of the mixture was adjusted to 8.5 with glacial acetic acid) wasadded to the reaction mixture and placed into a glass vial. The reactionvial was purged with nitrogen and stored at 37° C. for three days. Thepeptide was then loaded onto a prosorb column (Applied Biosystems) andabsorbed onto a PVDF membrane. After drying the PVDF membrane, 15 μl ofsolution A (280 μL methanol, 200 μL water, 65 μL 5 M sodium hydroxide,60 ethanethiol) was added to the membrane. The reaction was sealedtightly and incubated at 50° C. for 1 hour. After the reaction, thesample was sent out for Edman sequencing. The glass fiber filter used inthe Edman sequencing was pretreated with polybrene to reduce the loss ofpeptide per cycle. After drying in nitrogen, the PVDF membrane wasexcised and loaded onto a sequencer (Applied Biosystems 492 Protein P.E.Biosystems, Foster City, Calif., USA). The sequence was analyzed by theABI 610A data software. D,L-2-aminobutyric acid was commerciallypurchased from Sigma Aldrich and used as a standard.

MutD Cloning and Purification

A codon optimized sequence of mutD for E. coli was purchased fromIntegrated DNA Technologies (IDT, Coralville, Iowa). According tomanufacturer's specifications, mutD gene was cloned into the Xhol sitewithin the pET28B(+) expression vector (EMD Millipore, Billerica Mass.)providing an N-terminal His-tag. The ligation was transformed into E.coli DH5α and confirmed by sequencing. The plasmid was then transformedinto the E. coli BL21 expression strain. A fresh plate of E. coli BL21pET28B(+):mutD was restreaked onto LB plate containing 50 μg/mLkanamycin (kan) prior to induction. One colony from the plate wassuspended in 1 L of Terrific broth containing 50 μg/mL kan. Before theaddition of IPTG (250 μl of a 1M solution), the culture was shaken at37° C. until an OD₆₀₀ of 0.8 was reached. Following induction, theculture was incubated with shaking at 18° C. for approximately 16 hours.The culture was then spun at 4000 g for 30 minutes at 4° C. The pelletswere resuspended in 25 mL of lysis buffer (500 mM NaCl, 50 mM Tris-HCl,15 mM imidazole, 1 mM PMSF, 10% glycerol, at pH 7.5), before adding 500μl of the lysozyme solution (50 mg/mL). The solution was mixed andstored on ice for 30 min. The suspension was lysed using a sonicator atmedium setting for 10 minutes with 1 minute intervals, taking care tonot overheat the solution. The lysate was then centrifuged at 16,000 gand the supernatant was collected. 500 μl of Ni-NTA beads were added tothe supernatant and placed on a shaker for approximately 16 hours at 4°C. The Ni-NTA beads were collected by centrifugation at 3,000 RPM for 10minutes at 4° C. The beads were washed three times with 10× bead volumeof lysis wash buffer (500 mM NaCl, 50 mM Tris-HCl, 30 mM imidazole, 1 mMPMSF, at pH 7.5). After washing, the beads were eluted by resuspendingin 500 μL of lysis buffer containing 0.5 M Imidazole. The suspension wasplace on a shaker for 1 hour at 4° C. and the elution was repeated threetimes. The elutions were run on an SDS Page gel to determine purity ofthe MutD. The decarboxylase was further run on an FPLC. Proteinconcentrations were determined by Bradford assay (Sigma-Aldrich).

In vitro Decarboxylation

In vivo decarboxylation was performed as previously described²¹. Acontrol substrate, SFNSYTC was purchased from Peptide&Elephants. 1 mg/mLsolution of either SFNSYTC or mu1140-COOH in Tris-HCl buffer (pH 8.0)containing 3 mM DTT was prepared. The peptide solution (100 μL) wasincubated with MutD (30 μg/mL) for 1 to10 hours at 37° C. The sample wasdiluted 10-fold in 35% ACN containing 0.1% TFA before being loaded onthe RP-HPLC as previously described³¹. The masses of the isolatedfractions were determined by MALDI-TOF.

Lipid II Binding Assay

Lipid II was a kind gift from Eefjan Breukink and was resuspended in a1:1 Methanol:Chloroform solution. The lipid II binding assay using thinlayer chromatography (TLC) was done as previously described³⁹. Themobile solvent consisted of consisted of butanol:aceticacid:water:pyridine (15:3:12:10 [vol/vol/vol/vol]). A 0.2 mM solution ofmutacin 1140 or mu1140-COOH in 10 μL of solution A was mixed with lipidII (final 6.8 mM) for 1 hour in a sealed glass vial. This correspondedto a ratio 3:10 peptide:lipid II ratio. All of the reaction mixtures andthe appropriate controls were spotted (5 μL) 2 cm from the bottom of theplate. These spots define the origin of the plate. Lipid II and peptidealone were used as a control to demonstrate that the origins do notstain unless peptide and lipid II are added together. The mobile phasewas allowed to climb up the plate until it reached a centimeter from thetop. The plate was allowed to dry before staining with iodine.

All patents, patent applications, provisional applications, andpublications referred to or cited herein are incorporated by referencein their entirety, including all figures and tables, to the extent theyare not inconsistent with the explicit teachings of this specification.

Following are examples which illustrate procedures for practicing theinvention. These examples should not be construed as limiting. Allpercentages are by weight and all solvent mixture proportions are byvolume unless otherwise noted.

Example 1—Engineering a C-terminal Carboxyl Analog of an Epidermin GroupLantibiotic

A C-terminal carboxyl analog for the epidermin group of lantibiotics hasnot yet been characterized. Several structural analogs of mutacin 1140were identified when the formation of ring B was disrupted by a C(11)Amutation. The mutation interfered with the formation of the PTMsnormally found within mutacin 1140 and one of the peptide analogs hadnot undergone a C-terminal decarboxylation²⁹. This observation supportedthe basis for engineering S. mutans JH1140 to produce a mutacin 1140C-terminal carboxyl analog (mu1140-COOH). Deletion or insertionalmutagenesis of mutD was done using the IFDC2 gene replacement system(FIG. 5a )³⁰. A deferred antagonism assay, using the indicator strainMicrococcus luteus ATCC 10240, was performed on the S. mutansinsertional IFDC2:mutD and S. mutans ΔmutD mutant strains. Neithermutant strains had a clear zone of inhibition (FIG. 5b ), suggestingthat the mutants did not produce a product or that the product wasinactive. The culture broth of each mutant was extracted using the sameextraction method for wild-type mutacin 1140. These extracts were run onan HPLC, as previously described³¹. There was no observable product forthe ΔmutD strain. The ribosomal binding site (RBS) for mutP protease iswithin the 3′ end of the mutD gene. Careful consideration was made toleave the RBS site in the deletion strain. However, there may be otherelements important for the synthesis of downstream products that are notreadily apparent. Nevertheless, a single HPLC peak for IFDC2:mutD strainwas isolated and further characterized (FIG. 5c ). The IFDC2 cassette isunder the control of a constitutive lactose dehydrogenase (ldh) promoterand this promoter may facilitate the expression of the downstream genes.The purified product from this mutant was analyzed by MALDI-TOF massspectrometry and had a mass of 2310 Da. This mass corresponded to theexpected mass of a C-terminal carboxyl analog of mutacin 1140. A minimuminhibitory concentration (MIC) assay performed using mu1140-COOH analogagainst M. luteus revealed a 256-fold reduction in activity compared towild type mutacin 1140. Furthermore, the activity of mu1140-COOH againstStreptococcus pneumoniae ATCC 27336 was greater than 64 μg/mL, which ismore than a 128-fold reduction in activity (Table 7). The loss inactivity in the MIC assays further corroborates the lack of activityseen in the deferred antagonism assays. The reason for the lack ofactivity may be attributed to the presence of the carboxyl group or itcould indicate that the presence of the carboxyl group has disrupted theoccurrence of other PTMs found in mutacin 1140.

TABLE 7 Mass and activity of chemically modified analogs of mutacin 1140against select bacteria. MIC MIC Mutacin 1140 Expected Observed (μg/mL)(μg/mL) analog Mass (Da) Mass (Da) M. luteus S. pneumoniae Mutacin 11402264.63 2264.63 0.125 0.5 Mu1140 - COOH 2310.65 2310.63 32 >64 Mutacin1140 - 2325.72 2325.42 0.125 0.5 methylamine Mutacin 1140 - 2422.882421.97 0.25 2 diaminoheptane Mutacin 1140 - 2436.25 2435.80 1 8chlorophenylalanine Mutacin 1140 - 2470.69 2468.26 4 8 di-chlorophenylalanine

MALDI-TOF mass analysis can determine dehydrations due to an observablechange in mass. It cannot determine the formation of a lanthionine ringafter dehydration, since the PTM does not result in a change in mass ofthe peptide. Therefore, the formation of the lanthionine rings wasassessed by another method. A rapid and straight forward Edmansequencing method has been developed to distinguish between dehydratedresidues and dehydrated residues involved in lanthionine ringformation³². Dehydrated residues are first hydrated by sodiumborohydride before lanthionine ring derivatization by an organothiolcompound. This method can determine where the lanthionine rings areformed and the location of all the dehydrated residues, thus,elucidating the covalent structure of the lantibiotic peptide. Sodiumborohydride reduction of Dha and Dhb residues result in the formation ofalanine and 2-aminobutyric acid, respectively. Subsequent ethanethiolderivatization open the lanthionine rings, which form either thioethylcysteines (S-EC) or β-methylthioethyl cysteines (β-M-S-EC). The presenceof an S-EC or BM-S-EC residue at the amino acid positions 3, 8, 16, and19 indicate that the free thiols of upstream cysteines had reacted withtheir downstream dehydrated residues to form a lanthionine ring. Alanineand 2-aminobutyric acid were observed in positions 5 and 14,respectively (FIG. 6). These correspond to the reduction of the Dha5 andDhb14 residues. S-EC or BM-S-EC residues were observed in the expectedamino acid positions 3, 8, 16, and 19 (FIG. 6), confirming that they areinvolved in lanthionine ring formations. These data suggest that theloss of activity is not due to disruptions to the other PTM's and thatthe observed loss in bioactivity of the mu1140-COOH analog is due to thepresence of a C-terminal carboxyl group.

These results also demonstrate that the presence of the AviCys residueis not essential for the activity of a lantibiotic; however, the removalof the carboxyl group of the cysteine at the C-terminus is essential forthe activity of a lantibiotic.

Example 2—Restoration of Bioactivity of the C-Terminal Carboxyl Analogof an Epidermin Group Lantibiotic

Removal of the carboxyl group by MutD was attempted to confirm that thepresence of the C-terminal carboxyl group is responsible for the loss inbioactivity. A C-terminal histidine tag of MutD was constructed andoverexpressed in Escherichia coli. The purified product on an SDS-PAGEgel had the expected monomer size of 25 kDa; however, subsequentpurification through FPLC showed a product that was approximately 200kDa. 200 kDa mass is consistent with the formation of a homododecamer,as was previously reported for the epidermin decarboxylase EpiD²³. Todetermine if the enzyme was active, a reference peptide SFNSYTC wasincubated with MutD for one hour. A mass of 797.67 Da determined byMALDI-TOF was observed for the reference peptide compared to 843.69 Dafor the unreacted peptide, indicating that the decarboxylase was active(Table 8). mu1140-COOH analog was incubated with MutD for one or tenhours and showed no indication of a mass change by MALDI-TOF (Table 8).MutD was not capable of decarboxylating the mu1140-COOH analog.

TABLE 8 Mass of mu1140-COOH and control peptide after in vitrodecarboxylation with MutD. Mass before Expected mass Observed massSubstrate reaction (Da) (Da) (Da) SFNSYTC (1 hr) 843.69 797.67 797.67Mu1140-COOH 2310.37 2265.37 2310.63 (1 hr) Mu1140-COOH 2310.37 2265.372310.63 (10 hr)

Given that MutD was not capable of removing the C-terminal carboxylgroup, the C-terminal carboxyl group was chemically modified and testedto determine whether C-terminal substitutions could restore thebioactivity. EDC coupling of primary amines was used to cap theC-terminal carboxyl group (FIG. 2). None of the primary amines, testedon their own, had any activity against the indicator strains used inthis study. EDC coupling with methylamine, the smallest primary amine,was first attempted. The conjugation with methylamine yielded a productwith a mass of 2321 Da, indicating that the reaction occurred (Table 7).Based on the RP-HPLC spectra, the reaction did not yield any other sideproducts and the methylamine product was greater than 95% of thematerial. There was a small amount of unreacted mu1140-COOH analogeluting before the conjugated methylamine product. The methylamineconjugated mutacin 1140 analog had an MIC of 0.125 μg/mL against M.luteus, and 0.5 μg/mL MIC against S. pneumoniae. These values were thesame as the MIC values for native mutacin 1140. These results show thatcapping mu1140-COOH with a small primary amine restores activity to wildtype levels. This data also supports the notion that the presence of aC-terminal carboxyl group is responsible for the reduction in mu1140activity.

mu1140-COOH was then conjugated with diaminoheptane. The activity of thediaminoheptane conjugate was 0.5 μg/mL against M. luteus and 2.0 μg/mLagainst S. pneumoniae. The addition of two different chlorinatedaromatic rings with mu1140-COOH was also tested. The activity ofchlorophenylalanine conjugate was 1.0 μg/mL against M. luteus and 2.0μg/mL against S. pneumoniae. For the di-chlorophenylalanine conjugate,the activity was 1.0 μg/mL and 8.0 μg/mL against M. luteus and S.pneumoniae, respectively. All the conjugates restored the activity ofthe mu1140-COOH analog, supporting the synthesis of a library of analogsthat can be screened for novel applications.

Fluorescently labeled nisin, containing a C-terminal conjugate offluorescein, has been shown to form lipid II patches on the surfaceBacillus subtilis cells¹⁶. The binding of lipid II by an epidermin groupof lantibiotics has been shown in various in vitro assays³³; however,the bioactivity has never been visualized in vivo. This is due to thelack of amenable attachment site for a fluorophore. The free carboxylgroup on the mu1140-COOH analog provides one such site. A C-terminalfluorescein conjugate of mutacin 1140 was evaluated. The product had theexpected mass of 2623 Da and has the inhibitory activity in a deferredantagonism assay. For comparison, a C-terminal fluorescein conjugate ofnisin was also made as previously described¹⁶. As shown by Hasper etal., B. subtilis cells incubated with fluorescein labeled nisin showedlarge green patches on the cell surface (FIG. 3). The green patches havebeen attributed to lipid II abduction and sequestration by nisin fromits normal physiological location. The C-terminal fluorescein labeledmutacin 1140 produces a similar pattern of fluorescent patches asobserved by nisin (FIG. 3). This data supports in vitro data from theepidermin group of lantibiotics for lipid II binding and sequestrationand also demonstrates that this group of lantibiotics does sequesterlipid II from its normal physiological location for cell wall synthesis.

Example 3—The Loss of Activity for the C-Terminal Carboxyl Analog OfMutacin 1140

With the loss of activity associated with a free carboxyl group inmu1140-COOH, the basis for the loss in activity was studied. Themechanism of action for class I lantibiotics has been determined to bedue to lipid II binding³⁴⁻³⁶. In particular, rings A and B are believedto form a cage-like structure around the pyrophosphate moiety of lipidII³⁷. The latter half of the peptide is predicted to enhance binding andrecruit other lipid II-lantibiotic molecules into a large lipidII-lantibiotic complex¹⁶. Given that lipid II binding is done by theN-terminal rings A and B, it is likely that the loss of activity is dueto a loss in the lateral assembly function of the latter half of thepeptide. To test for this assumption, a series of experiments wereperformed to determine whether the mu1140-COOH would bind to lipid II orcompetitively block lipid II binding of nisin in vivo. A TLC plate assaywas used to determine if mu1140-COOH could bind to lipid II, as has beenpreviously reported for gallidermin³⁸. If lipid II migration on the TLCplate is impeded by binding to mutacin 1140 or mu1140-COOH, an iodinestained spot is observed at the origin. No stain was visible for mutacin1140, mu1140-COOH, and lipid II, when the compounds were spotted alone.A stained spot did appear at the origin when mutacin 1140 andmu1140-COOH were spotted with lipid II (FIG. 7). The mu1140-COOH analogmixed with lipid II showed a faint stain compared to lipid II mixed withwild-type mutacin 1140. The faint staining may be attributed to a weakerassociation of mu1140-COOH with lipid II than mutacin 1140. Ifmu1140-COOH is capable of binding to lipid II, but does not inhibit cellgrowth, it may provide resistance to a bacterium against mutacin 1140 ornisin. A competition MIC for mutacin 1140 and nisin was then performedagainst B. subtilis and M. luteus that were pretreated with mu1140-COOH.B. subtilis and M. luteus were pretreated with mu1140-COOH at 0.25 μg/mLand 0.125 μg/mL, respectively, before adding mutacin 1140. Mu1140-COOHcompetitively inhibited the activity of mutacin 1140 against B. subtilisand M. luteus by 4-fold (MIC 1.0 μg/mL and 0.5 μg/mL, respectively)(Table 9). When pretreated with 0.5 μg/mL of mu1140-COOH, the activityof nisin was competitively inhibited against B. subtilis and M. luteusby 4-fold and 2-fold, respectively (MIC 2.0 μg/mL and 1.0 μg/mL). Tofurther clarify a mechanism of action for the loss of activity in theMIC competition assay, confocal microscopy was used to observe nisinbinding as previously described³⁹. If mu1140-COOH is still binding tolipid II in vivo, it should competitively inhibit the binding offluorescein labeled nisin (FIG. 4). When the bacteria were pretreatedwith wild-type mutacin 1140 or mu1140-COOH, no typical in vivofluorescence pattern was observed for the fluorescein labeled nisin(FIG. 4). However, there was some association of fluorescein labelednisin with the bacteria, but nothing remotely similar to fluoresceinlabeled nisin on its own. This may be due to the weaker association ofmu1140-COOH to lipid II, which further corroborates the weak associationwith lipid II observed in the TLC assay. These results suggest that theloss in activity is not due to its inability to bind to the lipid IItarget, but due to its inability to form the large lantibiotic lipid IIcomplexes.

TABLE 9 Competition MICs of mutacin 1140 or nisin against B. subtilisand M. luteus preincubated with mu1140-COOH. B. subtilis B. subtilis M.Luteus M. Luteus MIC comp. MIC MIC comp. MIC Antibiotic (μg/mL) (μg/mL)(μg/mL) (μg/mL) Mutacin 0.25 1.0 0.125 0.5 1140 Nisin 0.5 2.0 0.5 1.0

Example 4—MUTACIN 1140 is Post-Translationally Modified DespiteDecarboxylation at the C-Terminus

Decarboxylation in mutacin 1140 is not needed for the other PTMmodifications; a fully modified analog of mutacin 1140 with a C-terminalcarboxyl group (mu1140-COOH) can be isolated and purified and thebactericidal activity can be restored by labeling the carboxyl groupwith a primary amine. Also, a fluorescein conjugated mutacin 1140 can besynthesized, enabling in vivo visualization of mutacin 1140 bound tolipid II target. Further, the loss of activity of the mu1140-COOH analogis likely due to the carboxyl group disrupting mutacin 1140 lipid IIcomplex formation and not due to the complete loss of lipid II binding.A lateral assembly mechanism that traps lipid II into a complex forbactericidal activity may be necessary, which is distinct for otherlipid II binding antibiotics, i.e. vancomycin⁴⁰. Furthermore, atgenerating a C-terminal labeled library of mutacin 1140 analogs mayexpand the lantibiotics' application or therapeutic use.

A variant of the epidermin group of lantibiotics with a C-terminalcarboxyl group is provided. Deletion of EpiD did not show activity;however, no discussion of an isolated product was mentioned²⁵. This ispresumably due to the lack of a product as was observed in the mutDdeletion strain. Increasing activity or developing new uses for existinglantibiotics has been a goal of many researchers in the field. This hasbeen achieved through a variety of methods, such as amino acidsubstitutions and the use of non-proteogenic amino acids^(26, 41-42).Semi-synthetic analogs of lantibiotics have been produced by chemicallymodifying lantibiotics⁴³⁻⁴⁴. The most common chemical modification oflantibiotics is through reactions with a free C-terminal carboxyl group.NVB302, is one such variant that has been chemically modified to have adiaminoheptane tail. This analog of actagardine has made it throughphase I clinical trials²⁷. Until the isolation of the mu1140-COOHanalog, the presence of an AviCys residue on the epidermin group oflantibiotics prevented further development of novel analogs. Theaddition of a chlorinated aromatic ring may confer additionalcharacteristics to mutacin 1140, as is seen in vancomycin analogs inwhich the vancomycin analog inhibited transglycosylase activity⁴⁵.Additionally, a diaminoheptane tail was conjugated to mutacin 1140,similar to NVB302. All of these analogs were bioactive.

Visualization of the bioactivity by the epidermin group of lantibioticswas limited to in vitro assays due the inability to conjugate adetectable label, for example, a fluorescent probe, to the antibiotic³³.The addition of a fluorescein label to mutacin 1140 allows the in vivovisualization of this class of lantibiotic in action. These data haveprovided new insights into the importance of decarboxylation forbioactivity of the epidermin group of lantibiotics. Class I lantibioticsare known to bind to lipid II by forming a cage around the pyrophosphateresidue using rings A and B (the lipid II binding domain)^(37, 46).Furthermore, the latter half of the peptide is believed to help in thelateral assembly of the lantibiotic-lipid II complexes to formislands^(16, 47). In nisin, these islands form a pore complex; however,the epidermin group primarily sequesters lipid II without forming apore, as has been reported in fluorescently labeled lipid II vesicleexperiments16, 48. Decarboxylation has been thought to be primarilyimportant for stability of the peptide by preventing carboxypeptidasesfrom degrading the lantibiotic⁴⁹. The loss of activity in themu1140-COOH analog is intriguing and is likely to be the result ofvarious factors; however, the data provided herein suggest that the lossin activity is primarily attributed to loss in lateral assemblyfunction. Microscopy studies show that mu1140-COOH can competitivelybind to lipid II against nisin. Nisin and mutacin 1140 have been shownnot to interact with each other, indicating that the decrease influorescence by the fluorescein labeled nisin analog was due tocompetition with the lipid II target³³. Additionally, the mu1140-COOHanalog was shown to have a protective function against wild-type mutacin1140 and nisin. This suggests that the lateral assembly activity iscrucial for bactericidal activity and the presence of the C-terminalcarboxyl group prevents mutacin 1140 forming a stable lipid II complex.

The influence of dehydrations and lanthionine ring formations onlantibiotic biosynthesis is well known⁵⁰⁻⁵¹. Yet, little is known on howother PTMs influence the biosynthesis of a functional lantibiotic.Studies have suggested that other PTM modifications, such as theN-terminal lactate of epilancin 15×, act independently of thedecarboxylation must occur before terminal ring formation due to areactive ene-thiol intermediate that promotes terminal ring formation²⁴.A crystal structure of MrsD has suggested that its active site cannotaccommodate a lanthionine ring²³. Furthermore, attempts atdecarboxylation of a lanthionine mimic proved futile²¹. The lack of invitro decarboxylation of mu1140-COOH shows that decarboxylation mustoccur before ring D formation suggesting that MutD cannot accommodatethe terminal lanthionine ring into its active site. Additionally, theisolation of the fully modified mu1140-COOH analog demonstrates thatterminal ring formation can occur regardless of the presence of acarboxyl group.

During in vivo synthesis of mutacin 1140, mutations that prevent ringformation or dehydrations within the lantibiotic may affect other PTMswithin the peptide²⁹. A better understanding of the role of each PTMwould promote the synthesis of novel analogs. The bioactivity of themu1140-COOH variant can be restored by capping the C-terminus with anamine (Table 7). The chemical synthesis of the AviCys residue iscumbersome⁵⁵⁻⁵⁶. However, chemical synthesis of this residue is notnecessary for the epidermin group of lantibiotics and that solid phasepeptide synthesis (SPPS) with differentially protected lanthionine canbe used to synthesize this class of lantibiotics. Thus, the inventiondescribed herein provides new possibilities for synthesizing novelanalogs of the epidermin group of lantibiotics. Furthermore, theimportance of decarboxylation for bioactivity is demonstrated.

Example 5—Site Directed Mutagenesis of Mutacin 1140 and its Effect onBactericidal Activity

Streptococcus mutans ATCC 55676 (wild-type) and the mutants that were atan OD₆₀₀ of 0.2 were spotted in triplicates on a pre-warmed THyex agarplate (150×15 mm) and allowed to dry. This assay was performed in thismanner to ensure that each sample had the same colony size for comparingzones of inhibition. The plate was incubated for 24 hours then placed inan oven at 50° C. for thirty minutes to kill the bacteria before theindicator strain was overlaid. Heat killing the bacteria prevents anyfurther antimicrobial compound production so that results seen are fromsingle day incubation. Zone diameter was measured in millimeters acrossthe plate and compared to wild-type (i.e. zone I for ATCC compared tozone I of Trp4Ala, etc.).

Lys2Ala, Leu6Ala, and Arg13Ala zones were statistically significantlyfrom the wild-type mutacin 1140 producing strain (FIG. 8). These mutantsled to a larger zone of inhibition and suggest that the bacterium isproducing more of the product or the antibiotic is more active. TheThr(Dhb)14Ala and Gly15Ala mutant had no change in activity relative towild-type strain.

Mutacin 1140 variants Lys2Ala, Leu6Ala, and Arg13Ala were purified forcomparing their bioactivity against wild-type mutacin 1140. MutantsLeu6Ala and Arg13Ala were two-fold more active against the indicatorbacterium Micrococcus lute us (Table 10). These mutants prove to bebeneficial in optimization of the antibacterial compound for thetreatment of infectious diseases. These mutants also provide greaterstability to the compound. For example, the Arg13Ala mutation makes themolecule less sensitive to gastric proteases.

The Lys2Ala mutant only had greater activity in the overlay assay, whichsuggests that this mutation leads to an increase in production of thecompound. This mutant would be useful for using the bacterium orengineered bacterium as a probiotic.

TABLE 10 Minimum inhibitory activity of mutant mutacin 1140 againstindicator strain Micrococcus luteus. Antibiotic MIC (μg/ml) Wild-typemutacin 1140 0.125 Mutant Lys2Ala 0.125 Mutant Leu6Ala 0.0625 MutantArg13Ala 0.0625

SEQ ID NO: 1 MKNSAAREAFKGANHPAGMVSEEELKALVGGNDVNPETTPATTSSWTCITAGVTVSASLCPTTKCTSRC SEQ ID NO: 2MKTEKKVLDELSLHASAKMGARDVESSMNADSTPVLASVAVSMELLPTAS VLYSDVAGCFKYSAKHHCSEQ ID NO: 3 MKNSKDILNNAIEEVSEKELMEVAGGKRGSGWIATITDDCPNSVFVCCSEQ ID NO: 4 MNKLNSNAVVSLNEVSDSELDTILGGNRWWQGVVPTVSYECRMNSWQHVF TCCSEQ ID NO: 5 MKEKNMKKNDTIELQLGKYLEDDMIELAEGDESHGGTTPATPAISILSAYISTNTCPTTKCTRAC SEQ ID NO: 6MSALAIEKSWKDVDLRDGATSHPAGLGFGELTFEDLREDRTIYAASSGWV CTLTIECGTVICACSEQ ID NO: 7 MSQEAIIRSWKDPFSRENSTQNPAGNPFSELKEAQMDKLVGAGDMEAACTFTLPGGGGVCTLTSECIC SEQ ID NO: 8MEAVKEKNDLFNLDVKVNAKESNDSGAEPRIASKFICTPGCAKTGSFNSY CC SEQ ID NO: 9MEAVKEKNELFDLDVKVNAKESNDSGAEPRIASKFLCTPGCAKTGSFNSY CC SEQ ID NO: 10MSNTQLLEVLGTETFDVQENLFTFDTTDTIVAESNDDPDTRFKSWSFCTP GCAKTGSFNSYCCSEQ ID NO: 11 MSNTQLLEVLGTETFDVQEDLFAFDTTDTTIVASNDDPDTRFKSWSLCTPGCARTGSFNSYCC SEQ ID NO: 12MRNDVLTLTNPMEEKELEQILGGGNGVLKTISHECNMNTWQFLFTCC SEQ ID NO: 13MPADILETRTSETEDLLDLDLSIGVEEITAGPAVTSWSLCTPGCTSPGGG SNCSFCC SEQ ID NO: 14TTPATTSSWTCITAGVTVSASLCPTTKCTSRC SEQ ID NO: 15STPVLASVAVSMELLPTASVLYSDVAGCFKYSAKHHC SEQ ID NO: 16KRGSGWIATITDDCPNSVFVCC SEQ ID NO: 17 NRWWQGVVPTVSYECRMNSWQHVFTCCSEQ ID NO: 18 TTPATPAISILSAYISTNTCPTTKCTRAC SEQ ID NO: 19SSGWVCTLTIECGTVICAC SEQ ID NO: 20 CTFTLPGGGGVCTLTSECIC SEQ ID NO: 21IASKFICTPGCAKTGSFNSYCC SEQ ID NO: 22 IASKFLCTPGCAKTGSFNSYCCSEQ ID NO: 23 FKSWSFCTPGCAKTGSFNSYCC SEQ ID NO: 24FKSWSLCTPGCARTGSFNSYCC SEQ ID NO: 25 GNGVLKTISHECNMNTWQFLFTCCSEQ ID NO: 26 VTSWSLCTPGCTSPGGGSNCSFCC

Example 6—Core Peptide Mutants of Mutacin 1140 and its Effect onBactericidal Activity

Mutacin 1140 has demonstrated activity against several resistant strainsof S. pneumoniae. Core peptide mutants of mutacin 1140 that haveimproved stability, superior pharmacokinetics, and higher activityagainst S. pneumoniae are provided. Particularly, core peptide mutantR13A of mutacin 1140 is provided as an alternative treatment option foran S. pneumoniae infection.

The formulations of the core peptide mutants, particularly, formulationssuitable for intravenous administration, are also disclosed. Suchformulations have improved toxicity profile and ability to treat an S.pneumoniae infection. Toxicological and pharmacokinetic evaluation ofR13A core mutant of mutacin 1140 following intravenous route ofadministration are described. R13A core mutant of mutacin 1140effectively treats a respiratory infection and bacteremia caused by S.pneumoniae.

The disruption of normal upper respiratory flora caused as a result ofconventional antibiotic treatment is the basis for the rampant spread ofS. pneumoniae. Conjugate vaccines protect against select serotypes, butthe niches occupied by these are being filled by one of the other 92serotypes of S. pneumoniae. The use of broad spectrum antibioticsactually increases the spread of antibiotic resistant S. pneumoniaeinfections in hospitals. Furthermore, the overuse of conventionalantibiotics has led to an emergence of resistant organisms by creatingreservoirs of resistant bacteria within the nasal passages that conferthe resistance to S. pneumoniae. There are more than 4 million S.pneumoniae infections each year. Pneumococcal ear infections (otitismedia) make up 1.5 million of these cases, and they are a direct resultof conventional antibiotic use (CDC, Antibiotic Resistance Report 2013).At least 400,000 hospitalizations are estimated from pneumococcalpneumonia each year in the United States (CDC). Approximately thirtypercent of S. pneumoniae infections are resistant to one or moreantibiotics. These infections lead to excessive hospital stays anddeath. Patients with pneumococcal pneumonia have a high incidence ofbacteremia (˜30%) leading to death in about 6% of the cases. Despite thepositive outcome of the conjugate vaccine, a need exists for developingalternative approaches to lessen the burden associated with pneumococcalpneumonia. The increasing tolerance of pneumococcal infections topenicillin and vancomycin is alarming. Certain embodiments of theinvention provide new antibiotics with potent activity against S.pneumoniae that can be added to the dwindling arsenal of treatmentoptions.

Mutacin is a peptide belonging to class I lantibiotics and is naturallyproduced by a strain of the common oral bacterium, Streptococcus mutans.This compound has demonstrated activity against several Gram-positivepathogens, including oxacillin- and vancomycin-resistant Staphylococcusaureus and vancomycin-resistant E. faecalis. The post-translationalincorporation of lanthionine rings confers proteolytic stability, whichshould enhance its stability and use in animals. However, mutacin 1140like other lantibiotics has a short half-life in animals. The inventionprovides core peptide mutants of mutacin 1140 that have potent activityagainst S. pneumoniae and have the pharmacokinetic and pharmacodynamicparameters supporting its potential usefulness in treating apneumococcal infection in animals.

Mode of action of mutacin: Sequential subculturing of S. aureus and S.pneumoniae in sub-inhibitory concentrations of mutacin resulted in onlya three-fold increase in the minimum inhibitory concentration,suggesting that this antibiotic is a good candidate as a therapeuticagent against infection. Mutacin functions, much like vancomycin, bybinding to lipid II, which is essential for cell-wall formation.Although vancomycin shares the same target, it uses a differentmechanism to inhibit cell wall synthesis. Vancomycin binds to adifferent region on lipid II, which are the terminal D-Ala-D-Alaresidues of the peptide. This difference in activity possiblycontributes to mutacin's impressive ability to kill vancomycin-resistantstrains of bacteria. Mutacin kills bacteria by sequestering theessential cell wall biosynthetic molecule lipid II into domains awayfrom the sites that are required for cell wall synthesis. Lipid II isessential for the transport of cell wall subunits across the bacterialcytoplasmic membrane. This highly dynamic molecule is present in alleubacteria in relatively small amounts. Lipid II is assembled on thecytoplasmic side of the bacterial membrane and carries one completepeptidoglycan subunit (GlcNAc-MurNAc-pentapeptide) across the plasmamembrane. A novel lipid II-binding motif for mutacin-relatedlantibiotics has been characterized by NMR, in which the N-terminalportion of the lantibiotic, lanthionine rings A and B, interact with thepyrophosphate, peptidoglycan MurNAc, and first isoprene of lipid II.Mutacin activity is also attributed to bacterial membrane disruption. Noprotein receptor is required for the bactericidal activity of this classof antibiotics, and therefore, it cannot easily be overcome via geneticadaptation. This unique mechanism of action for mutacin and similarlantibiotics support the long-term value of a new antibiotic.

Therapeutic activity of mutacin: The solubility of pure native mutacinin aqueous saline solution exceeds 0.5 mg/mL, which is well above itslow inhibitory concentration against Gram-positive bacteria. Mutacin hasa broad spectrum of activity against Gram-positive bacteria. Inparticular, mutacin has a submicromolar activity against S. pneumoniae.Furthermore, mutacin has been shown to be active against oxacillin- andvancomycin-resistant S. aureus, as well as vancomycin-resistant E.faecalis. No activity was observed against Gram-negative bacteria oryeast consistent with previous lantibiotic studies. The level ofactivity varied by more than 128-fold between species of Gram-positivebacteria, which may promote its use for treating S. pneumoniaeinfections. Disease associated with S. pneumoniae is usually attributedto the disruption of host flora. The lack of activity againstGram-negative bacteria and the increased susceptibility of pneumococcalbacteria relative to other Gram-positive bacteria may promote thereestablishment of the host's protective flora during treatment. Resultsof acute toxicity studies in rodents support the further development ofanalogs of mutacin for the treatment of Gram-positive infections. Mouseand rat models were tested by bolus intravenous injection of mutacin innormal saline. Both models readily tolerated doses up to 25 mg/kg bodyweight. Time-kill studies have been performed by using strains ofmedically important Gram-positive species, S. aureus and E. faecalis.The results of time-kill investigations showed that mutacin exhibited arapid initial killing against multidrug resistant S. aureus, whereasbacteriostatic activity was observed against a vancomycin resistantstrain of E. faecalis. This is very similar to the activity seen withvancomycin. However, the native mutacin 1140 has a short half-life andis rapidly cleared from the blood, which prevents its use anddevelopment as an effective treatment option.

Several mutacin core peptide mutants were tested for their ability toinhibit several clinical strains of S. pneumoniae, while also havingsuperior pharmacokinetic and stability properties. The pharmacokineticsand efficacy of certain mutacin core peptide mutants were tested toprovide treatment options for an S. pneumoniae infection.

Example 7—Formulation, Toxicological, and Pharmacokinetic Evaluation ofthe R13A Core Peptide Mutant of Mutacin 1140 Following IntravenousAdministration

R13A core peptide mutant of mutacin 1140 was tested as a noveltherapeutic for pneumococcal infections. As described above, nativemutacin has several attributes to serve as a successful therapeuticagent, but its low peak plasma concentration and rapid clearance fromblood prevents its development as an effective therapy. Core peptidemutants of mutacin 1140 that have superior properties compared to nativemutacin are provided. Also, formulations of R13A that improvespharmacokinetic properties of R13A are also provided.

FIG. 11 illustrates the bioactivity of various core peptide mutants ofmutacin 1130. The activity of the core mutant peptides is expressed as aratio of the area of the zone of inhibition of each mutant strain to thearea of the zone of inhibition of the wild-type strain against indicatorstrain of Micrococcus luteus. A value of one denotes similar activity towild-type, but values greater than one shows higher activity for themutants. The deferred antagonism assay is an overlay assay of theindicator strain onto plates containing heat killed colonies of themutacin producer strain after growing overnight. Core peptide mutantshaving inhibitory activity were isolated and the purified products werefurther evaluated by a standard minimum inhibitory concentration (MIC)assay. MICs for some of the core peptide mutants of mutacin 1140 areprovided in Table 11.

TABLE 11 Bioactivity of core peptide mutants of mutacin 1140. C.accolens KPL 1818 and C. accolens ATCC 49725 were cultured in THyexbroth supplemented with 1% Tween 80 to promote the bacterial growth.MICs of wild-type mutacin 1140 were done against M. luteus, S. aureus,and S. pneumoniae with or without 1% Tween 80 at the same time and therewas no difference in activities. MICs and MCLs are presented inmicrograms per milliliter. Mutacin M. luteus S. aureus C. accolens C.accolens S. pneumonia 1140 ATCC 10240 ATCC 25923 ATCC 49725 KPL 1818ATCC 27336 Analogs MIC MLC MIC MLC MIC MLC MIC MLC MIC MLC Wild- 0.06250.0625 8 8 0.125 0.125 1 2 0.5 0.5 Type Mutacin Mutations within ring AS5T 4 4 >32 >32 >4 >4 >4 >4 2 2 S5G 0.125 0.125 8 8 0.5 0.5 0.5 0.50.0078 0.0078 Mutations within hinge region R13A 0.0625 0.0625 16 160.25 0.5 1 1 0.125 0.125 T14G 0.0625 0.0625 >32 >32 0.0625 0.0625 0.250.5 0.5 0.5 T14A 0.125 0.125 8 16 0.25 0.25 0.5 2 0.5 0.5 G15A* 0.1250.125 16 16 1 1 1 2 0.5 0.5 R13A: 0.25 0.5 32 32 0.5 0.5 0.5 0.5 2 2T14A A12T: 0.25 0.25 >32 >32 0.125 0.25 0.5 1 2 2 T14G Mutations withinboth ring A and hinge region S5G: 0.125 0.125 >32 >32 4 4 — — 4 4 T14AS5A: 0.0625 0.125 32 32 0.25 0.25 0.5 1 0.25 0.25 T14G S5G: 0.5 0.5 8 161 1 0.5 1 2 2 T14G S5A: 0.125 0.25 16 32 0.25 0.25 0.5 1 0.125 0.125A12S dehydrated S5A: 0.125 0.25 16 16 — — — — 0.125 0.125 A12S un-dehydrated S5A: 0.125 0.25 >32 >32 2 2 2 4 0.5 0.5 R13S S5G: 216 >32 >32 >4 >4 >4 >4 8 >16 R13A: T14A

Selective activity and stability of analogs: Several core peptidemutants are provided that have improved activity against our ATCC strainof S. pneumoniae. These core peptide mutants were also selected based onthe absence of a dehydrated residue Dha/Dhb or the proteolyticallysusceptible arginine residue. While the S5G, S5A:T14G, S5A, T14A, andT14G had low micromolar activity against one or more of the clinical S.pneumoniae isolates, the R13A analog had superior activity against moststrains compared to wild-type mutacin (Table 12). Although the analogS5G had improved activity against S. pneumoniae ATCC 27336 compared tonative mutacin and R13A, the S5G had less activity against all othertested strains of S. pneumoniae. Further supporting data for thedevelopment of the R13A analog are: 1) the activity of R13A wasunchanged when tested in the presence of 50% serum, 2) In a trypsinstability screen (native mutacin and the analogs were spotted on a THyexplate overlayed with M. luteus before and after treatment with 0.5 mg/mLof trypsin at 37° C. for 30 minutes), R13A has improved stabilitycompared to native mutacin, T14A, and S5A:T14G (FIG. 13) The solubilityof the R13A analog in saline is greater than 2 mg/mL, which is afour-fold increase in solubility of native mutacin. 4) The R13A mutationprevents feedback inhibition of mutacin 1140 production, leading to a100% increase in yield from the culture liquor. The improved stabilityof R13A in the presence of trypsin protease is encouraging when comparedto native mutacin, since trypsin-like proteases are abundantly presentin the blood. They are important for immunoregulation and bloodcoagulation. The observed increase in stability of the R13A analog willlikely be advantageous in an animal model.

TABLE 12 MICs of mutacin analogs against clinical S. pneumoniaeisolates. S. pneumoniae isolates required the addition of 50% blood forgrowth in the bioassay. MICs and MLCs are presented in micrograms permilliliter. S. pneumoniae S. pneumoniae S. pneumoniae S. pneumoniae S.pneumoniae AI8 AI11 AI14 AI16 AI17 Mutacin 1140 1 1 0.125 1 0.25 R13A0.5 0.5 0.25 0.5 0.25 S5G >8 8 >8 >8 2 S5A:T14G >8 >8 >8 >8 8 S5A 8 4 >84 4 T14A 8 4 2 8 8 T14G 8 2 1 8 8

Pharmacokinetic comparison of native mutacin and analogs: Murine modelsof S. pneumoniae infection are easy to use and commonly employed tostudy efficacy of antibiotic treatments. Murine models of S. pneumoniaeinfection provide an easy and reliable platform to test the efficacy ofcandidate drugs for treatment. The efficacy of the novel formulation ofR13A was evaluated in a mouse model of pneumococcal pneumonia.

The compounds were dissolved in normal saline for all pharmacokineticstudies. An extraction protocol was developed in vitro by the additionof each compound to commercially bought BALB/c mouse serum. Thecompounds were extracted by the addition of 70% ACN containing 0.1%TFA/30% methanol containing 0.4% formic acid. The extract was thenanalyzed by LC-MS using a TSQ Vantage Triple Quad mass spectrometer. Astandard curve was generated for each compound using known dilutions ofthe compounds in serum, which was then extracted. The R-squared valuesfor each standard exceeded 0.98. BALB/c mice were used to evaluatepharmacokinetics of the compounds in vivo. A 2.5 mg/kg i. v. dose wasevaluated for native mutacin, R13A, S5G, and double mutant S5A:T14G(FIG. 12A). A polynomial second order was used to estimate the peakplasma concentration and half-life of the R13A. The peak plasmaconcentration of R13A was 1700 ng/mL and the estimated half-life was 70minutes. At 70 minutes, native mutacin and other mutant analogs wereabout to be cleared from the blood. Interestingly, the double mutantS5A:T14G performed the worst. This data in combination with the S5Gdata, suggests that the Dhb residue normally at position 14 is importantfor peptide stability. Possibly, the Dhb14 residue may protect thepeptide from trypsin-like proteases in the blood. As seen in the invitro studies, at 125 ng/mL, the R13A analog reduces the number of S.pneumonia by 2-logs (FIG. 12B). Thus, taking the dynamics of cell deathinto account and the pharmacokinetics of the R13A, the R13A in serum isabove the inhibitory concentration for S. pneumonia long enough to killthe pathogen. R13A is more stable and more active against S. pneumoniaestrains and is pharmacokinetically superior to native mutacin.

Example 8—Formulations of Antimicrobial Peptides

Antimicrobial peptides can have poor solubility. Solubilizing/surfactantagents are used to meet the strict USP requirements for purity andclarity of injectable formulations. R13A is soluble at 2 mg/ml in salinesolution. To further improve the solubility, solubilizing agents can beused. The solid stock of R13A can be prepared by lyophilization, storedat 4° C., and reconstituted as needed. In certain embodiments, theformulation comprises 50 mg of R13A, 50 mg of fructose (stabilizer), 269mg of mannitol (bulking agent), 125 mg of polysorbate-80 (solubilizingagent), and 6 mg of tartaric acid (buffer). 50 mg of solid stock can bereconstituted in 1-2 mL of water as needed (a 16-8 mg/mL of active agentstock). In further embodiments, the formulations of R13A furthercomprise excipients such as β-cyclodextrins, Tween 20, PEG 400,demethylacetamide. Such formulations can contain up to 8 mg/mL R13A.Also, 5-20% ethanol can be used to reconstitute the solid stock.

It should be understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication and the scope of the appended claims. In addition, anyelements or limitations of any invention or embodiment thereof disclosedherein can be combined with any and/or all other elements or limitations(individually or in any combination) or any other invention orembodiment thereof disclosed herein, and all such combinations arecontemplated within the scope of the invention without limitationthereto.

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We claim:
 1. A modified lantibiotic selected from mutacin 1140comprising SEQ ID NO: 24 and mutacin B-Ny266 comprising SEQ ID NO: 23,said modified lantibiotic having a free carboxy group at the C-terminus,wherein: a) mutacin 1140 has mutations at: i) one or more of Leu6 andArg13 to an amino acid selected from alanine, glycine, valine, leucineor isoleucine, ii) one or more of Leu6 and Arg13 to alanine, or iii)Lys2 to an amino acid selected from alanine, glycine, valine, leucine orisoleucine, or b) mutacin B-Ny266 has mutations at: i) one or more ofPhe6 and Lys13 to an amino acid selected from alanine, glycine, valine,leucine or isoleucine, ii) one or more of Phe6 and Lys13 to alanine, oriii) Lys2 to an amino acid selected from alanine, glycine, valine,leucine or isoleucine; or c) mutacin 1140 or mutacin B-Ny266 is mutatedat the following amino acid positions: Original amino acid(s)Replacement amino Amino acid position(s) (respectively) acid(s),respectively 5 Serine Glycine 5 Serine Threonine 5 Serine Glutamate 5Serine Alanine 13  Arginine Alanine 14  Threonine Glycine 14  ThreonineAlanine 15  Glycine Alanine 12  Alanine Threonine 4 Tryptophan Serine 6Leucine Serine 12, 14  Alanine and Threonine Threonine and Glycine 13,14  Arginine and Threonine Alanine and Alanine 14, 15  Threonine andGlycine Alanine and Alanine 5, 14 Serine and Threonine Glycine andGlycine 5, 14 Serine and Threonine Alanine and Glycine 5, 14 Serine andThreonine Threonine and Glycine 5, 14 Serine and Threonine Alanine andSerine 5, 14 Serine and Threonine Alanine and Alanine 5, 14 Serine andThreonine Glycine and Alanine 5, 14 Serine and Threonine Glutamate andAlanine 5, 14 Serine and Threonine Threonine and Alanine 5, 12 Serineand Alanine Alanine and Serine 5, 13 Serine and Arginine Alanine andSerine 13, 14 and 15 Arginine, Threonine and Alanine, Alanine andGlycine Alanine  5, 13 and 14 Serine, Arginine and Glcyine, Alanine andThreonine Alanine  4, 5 and 14 Tryptophan, Serine and Serine, Alanineand Threonine Alanine  5, 6 and 14 Serine, Leucine and Alanine, Serineand Threonine Alanine  5, 12 and 14 Serine, Alanine and Alanine, Serineand Threonine Alanine  5, 13 and 14 Serine, Arginine and Alanine, Serineand Threonine Alanine 12, 13 and 14 Alanine, Arginine and Glycine,Glycine and Threonine Glycine.


2. The modified lantibiotic according to claim 1, wherein said modifiedlantibiotic is: a) mutacin 1140 having mutations at: i) one or more ofLeu6 and Arg13 to an amino acid selected from alanine, glycine, valine,leucine or isoleucine, ii) one or more of Leu6 and Arg13 to alanine, oriii) Lys2 to an amino acid selected from alanine, glycine, valine,leucine or isoleucine, or b) mutacin B-Ny266 having mutations at: i) oneor more of Phe6 and Lys13 to an amino acid selected from alanine,glycine, valine, leucine or isoleucine, ii) one or more of Phe6 andLys13 to alanine, or iii) Lys2 to an amino acid selected from alanine,glycine, valine, leucine or isoleucine.
 3. The modified lantibioticaccording to claim 2, wherein the mutant mutacin 1140 has an amino acidmutation at position 2 or
 13. 4. The modified lantibiotic according toclaim 3, wherein mutacin 1140 has an amino acid mutation at position 2selected from alanine, glycine, valine, leucine or isoleucine.
 5. Themodified lantibiotic according to claim 4, wherein mutacin 1140 ismutated to have an alanine at position
 2. 6. The modified lantibioticaccording to claim 3, wherein mutacin 1140 has an amino acid mutation atposition 13 selected from alanine, glycine, valine, leucine orisoleucine.
 7. The modified lantibiotic according to claim 6, whereinmutacin 1140 is mutated to have an alanine at position
 13. 8. Apharmaceutical composition comprising mutacin modified lantibioticaccording to claim 1 and pharmaceutically acceptable carrier orexcipient.
 9. A lantibiotic peptide comprising SEQ ID NO: 24 having anamino acid mutation at position 2 in which the lysine residue issubstituted with an alanine and having free C-terminal carboxy group ora decarboxylated C-terminal amino acid.
 10. The lantibiotic peptideaccording to claim 9, wherein the mutant mutated mutacin has a freeC-terminal carboxy group.
 11. The lantibiotic peptide according to claim9, wherein the mutant mutated mutacin is decarboxylated at theC-terminal amino acid.
 12. A pharmaceutical composition comprisingmutacin lantibiotic peptide according to claim 9 and pharmaceuticallyacceptable carrier or excipient.
 13. A mutated mutacin selected frommutacin 1140 comprising SEQ ID NO: 24 and mutacin B-Ny266 comprising SEQID NO: 23, said mutated mutacin being produced by a bacteriumgenetically modified to inactivate a gene that encodes a decarboxylaseenzyme that decarboxylates the cysteine at the C-terminus of said mutantmutacin, wherein: a) mutacin 1140 has mutations at: i) one or more ofLeu6 and Arg13 to an amino acid selected from alanine, glycine, valine,leucine or isoleucine, ii) one or more of Leu6 and Arg13 to alanine, oriii) Lys2 to an amino acid selected from alanine, glycine, valine,leucine or isoleucine, or b) mutacin B-Ny266 has mutations at: i) one ormore of Phe6 and Lys13 to an amino acid selected from alanine, glycine,valine, leucine or isoleucine, ii) one or more of Phe6 and Lys13 toalanine, or iii) Lys2 to an amino acid selected from alanine, glycine,valine, leucine or isoleucine; or c) mutacin 1140 or mutacin B-Ny266 ismutated at the following amino acid positions: Amino acid Original aminoacid(s) Replacement amino position(s) (respectively) acid(s),respectively  5 Serine Glycine  5 Serine Threonine  5 Serine Glutamate 5 Serine Alanine 13 Arginine Alanine 14 Threonine Glycine 14 ThreonineAlanine 15 Glycine Alanine 12 Alanine Threonine  4 Tryptophan Serine  6Leucine Serine 12, 14 Alanine and Threonine Threonine and Glycine 13, 14Arginine and Threonine Alanine and Alanine 14, 15 Threonine and GlycineAlanine and Alanine  5, 14 Serine and Threonine Glycine and Glycine  5,14 Serine and Threonine Alanine and Glycine  5, 14 Serine and ThreonineThreonine and Glycine  5, 14 Serine and Threonine Alanine and Serine  5,14 Serine and Threonine Alanine and Alanine  5, 14 Serine and ThreonineGlycine and Alanine  5, 14 Serine and Threonine Glutamate and Alanine 5, 14 Serine and Threonine Threonine and Alanine  5, 12 Serine andAlanine Alanine and Serine  5, 13 Serine and Arginine Alanine and Serine13, 14 and 15 Arginine, Threonine and Alanine, Alanine and GlycineAlanine  5, 13 and 14 Serine, Arginine and Glycine, Alanine andThreonine Alanine  4, 5 and 14 Tryptophan, Serine and Serine, Alanineand Threonine Alanine  5, 6 and 14 Serine, Leucine and Alanine, Serineand Threonine Alanine  5, 12 and 14 Serine, Alanine and Alanine, Serineand Threonine Alanine  5, 13 and 14 Serine, Arginine and Alanine, Serineand Threonine Alanine 12, 13 and 14 Alanine, Arginine and Glycine,Glycine and Threonine Glycine.


14. The mutated mutacin according to claim 13, wherein said mutantmutated mutacin is: a) mutacin 1140 having mutations at: i) one or moreof Leu6 and Arg13 to an amino acid selected from alanine, glycine,valine, leucine or isoleucine, ii) one or more of Leu6 and Arg13 toalanine, or iii) Lys2 to an amino acid selected from alanine, glycine,valine, leucine or isoleucine, or b) a mutacin B-Ny266 having mutationsat: i) one or more of Phe6 and Lys13 to an amino acid selected fromalanine, glycine, valine, leucine or isoleucine, ii) one or more of Phe6and Lys13 to alanine, or iii) Lys2 to an amino acid selected fromalanine, glycine, valine, leucine or isoleucine.
 15. The mutated mutacinaccording to claim 13, wherein the mutated mutacin 1140 has an aminoacid mutation at position 2 or
 13. 16. The mutated mutacin according toclaim 15, wherein the mutated mutacin 1140 has an amino acid mutation atposition 2 selected from alanine, glycine, valine, leucine orisoleucine.
 17. The mutated mutacin according to claim 16, wherein themutated mutacin 1140 is mutated to have an alanine at position
 2. 18.The mutated mutacin according to claim 15, wherein the mutated mutacin1140 has an amino acid mutation at position 13 selected from alanine,glycine, valine, leucine or isoleucine.
 19. The mutated mutacinaccording to claim 18, wherein the mutated mutacin 1140 is mutated tohave an alanine at position
 13. 20. A pharmaceutical compositioncomprising a mutated mutacin according to claim 13 and pharmaceuticallyacceptable carrier or excipient.